Herpes simplex virus 1 harboring poly(T) DNA sequences as a key ligand for AIM2 inflammasome activation and host defense

Domain: biology
Published: 2026-04-13
Editorial outcome: minor_revision

Abstract
Herpes simplex virus type 1 (HSV-1) infection remains a major global health challenge, yet the
mechanisms underlying strain-specific innate immune responses are poorly understood. Here,
we show that distinct HSV-1 strains differentially activate the absent in melanoma 2 (AIM2)
inflammasome. The HF strain robustly induces AIM2-dependent inflammasome activation,
whereas the F and KOS strains elicit minimal responses despite comparable infection efficiency.
We demonstrate that this difference is driven by viral genomic features rather than replication
capacity. Genomic analyses identify a poly(T) DNA sequence within the UL25–UL26 intergenic
region that is enriched in the HF strain. Deletion of a 14-mer poly(T) sequence markedly impairs
inflammasome activation, cytokine release, and host protection in vivo, whereas introduction of a
poly(T) tract into the F strain is sufficient to confer AIM2 activation and enhanced host defense.
Furthermore, poly(T)-mediated AIM2 activation is length-dependent, conserved in human
macrophages, and requires a cGAS–STING–IRF1 licensing axis. Together, these findings identify
viral poly(T) DNA as a key determinant of strain-specific AIM2 inflammasome activation and
reveal how viral genomic variation shapes innate immune recognition.

Introduction
Herpes simplex virus type 1 (HSV-1) infection poses a significant global public health challenge,
affecting approximately 67% of the global population. With a genome spanning 152 kbp of linear
double-stranded DNA (dsDNA), HSV-1 encodes a diverse repertoire of at least 84 viral proteins.
Although antiviral therapies can suppress viral replication and alleviate symptoms, HSV-1
establishes lifelong latency and periodically reactivates, underscoring the absence of curative
strategies and the need for a deeper understanding of virus–host interactions that govern immune
control and pathogenesis1, 2. Upon infiltration into host cells, HSV-1 triggers the prompt activation
of innate immunity and subsequent production of inflammatory cytokines and cell death, crucial
for orchestrating a natural antiviral immune response essential for viral clearance and preventing
prolonged infection.

Among innate immune defense mechanisms, inflammasome activation plays a pivotal role in
detecting intracellular pathogens and initiating inflammatory cell death. Inflammasomes are
multiprotein complexes that assemble in response to pathogen-associated molecular patterns
(PAMPs) or damage-associated molecular patterns (DAMPs), leading to caspase-1 activation,
maturation of interleukin-1β (IL-1β) and interleukin-18 (IL-18), and induction of pyroptosis3, 4, 5.
These responses eliminate infected cells and amplify antiviral immunity. Several cytosolic
inflammasome sensors have been characterized, including NOD-like receptor family pyrin domain
containing 3 (NLRP3), NOD-like receptor family CARD domain-containing protein 4 (NLRC4), and
absent in melanoma 2 (AIM2), each responding to distinct molecular triggers. Notably, AIM2
directly binds cytosolic dsDNA through its hematopoietic interferon-inducible nuclear domain,
forming an inflammasome complex that is essential for host defense against intracellular bacteria
and DNA viruses6, 7, 8, 9, 10, 11, 12.

HSV-1 research has revealed a complex interplay of evolutionary nuances and genetic diversity,
intricately influencing its interactions with the host immune system1, 2. A notable aspect of this
complexity lies in the differential activation of the AIM2 inflammasome by distinct HSV-1 strains.
For example, while the F strain exhibits nuanced control over AIM2 inflammasome activation
through the actions of viral protein 22 (VP22)13, 14, the HF strain robustly stimulates AIM2
inflammasome activation and inflammatory cell death7, 11. This distinction underscores the
intricate interplay between viral genetics and host immune response, shedding light on the
multifaceted dynamics governing HSV-1 pathogenesis. To date, no hypotheses or claims have
been proposed to explain these strain-specific differences, suggesting a need for further research
to deepen our understanding of the interactions between HSV-1 and the host immune system.

To elucidate the mechanisms of AIM2 inflammasome activation by distinct HSV-1 strains, we
screened specific DNA ligands and identified 14-base pair (bp) poly(T) DNA sequences as key
ligands of HSV-1 for AIM2 inflammasome activation and host defense. These findings advance
our understanding of innate immunity, inflammasomes, and HSV-1 pathogenesis, paving the way
for the development of novel therapeutics against HSV-1.

Results
HSV-1 HF strain selectively triggers AIM2 inflammasome activation and inflammatory cell
death.
To elucidate HSV-1 involvement in apoptosis-associated speck-like protein containing a CARD
(ASC) inflammasome activation, we performed immunofluorescence staining using antibodies
against ASC and tubulin. ASC speck-positive cells were observed following infection with three
distinct HSV-1 strains: HF, F, and KOS. Notably, infection by the HF strain led to ASC speck
formation, whereas the F and KOS strains did not induce this phenomenon (Fig. 1a). Furthermore,
HSV-1 infection prompted caspase-1 cleavage (Fig. 1b), release of inflammasome-driven
cytokines, IL-1β (Fig. 1c) and IL-18 (Fig. 1d), as well as cell death (Fig. 1e, f), in an HF strain-
dependent manner.

Importantly, HSV-1 HF, F, and KOS strains exhibited comparable viral protein expression kinetics
in both HaCaT cells and primary wild-type (WT) or Aim2−/− bone marrow-derived macrophages
(BMDMs), indicating similar infection efficiency across strains (Supplementary Fig. S1a, b).
Subsequently, we extended our observations to in vivo models of HSV-1 infection with the
different strains. While WT mice succumbed to F and KOS strain infections within 13 days,
approximately 80% of WT mice infected with the HF strain survived (Fig. 1g). Furthermore, plaque
assays of lung tissue homogenates from infected mice revealed significantly lower viral titers in
the HF strain compared with those in the F and KOS strains (Fig. 1h). Lung lysates from infected
WT mice exhibited caspase-1 cleavage (Fig. 1i), and IL-1β (Fig. 1j) and IL-18 (Fig. 1k) were
detected in bronchoalveolar lavage fluid (BALF) in an HF strain-dependent manner. These results
indicate that inflammasome activation and cell death are specifically induced by infection with the
HF strain of HSV-1.

To investigate whether cells infected with the HF strain of HSV-1 engage the inflammasome
sensor, WT BMDMs and BMDMs singly deficient in several major inflammasome sensors were
infected with HSV-1. Infection with the HF strain of HSV-1 induced AIM2-dependent cleavage of
caspase-1 and cell death, independent of NLRP3 and NLRC4 (Supplementary Fig. S2a–i),
suggesting that AIM2 serves as an inflammasome sensor for the HF strain of HSV-1. To
determine AIM2 involvement in the F and KOS strains of HSV-1, we infected WT and AIM2-
deficient BMDMs with HF, F, and KOS strains. Infection with the HF strain induced AIM2-
dependent cell death, whereas the F and KOS strains did not induce cell death in either WT or
AIM2-deficient BMDMs (Fig. 2a, b).

In addition to inflammasome activation, various pathogens and sterile triggers have been
identified to induce programmed cell death pathways, including pyroptosis, apoptosis, and
necroptosis7, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. Thus, our aim was to perform a biochemical
characterization of HSV-1-induced cell death. In WT BMDMs, infection with the HF strain of HSV-
1 induced activation of caspase-1 and GSDMD (pyroptotic), caspase-8, -3, and -7 (apoptotic),
and MLKL (necroptotic) in an AIM2-dependent manner, whereas infection with the F and KOS
strains did not activate these pathways (Fig. 2c, d, e). As the HF strain of HSV-1 induced activation
of multiple cell death molecules, we investigated the relative contribution of each of them using a
genetic approach. Despite reports of multiple caspases regulating cell death in different contexts17,
29, 30, 31, immortalized BMDMs (iBMDMs) lacking Casp1, Casp3, Casp6, Casp7, or Casp9 showed
similar dynamics of cell death compared with WT iBMDMs (Supplementary Fig. S3a, c, d). Loss
of the pyroptotic executioner GSDMD and GSDME and necroptotic executioner MLKL did not
protect cells against HF strain infection (Supplementary Fig. S3b, e, f). In contrast, Ripk3-deficient
iBMDMs showed reduced cell death compared with WT iBMDMs, and Ripk3/Casp8 double-
knockout iBMDMs completely abrogated cell death against HF strain infection (Supplementary
Fig. S3b, e, f), suggesting that RIPK3 and caspase-8 are key executioner molecules in this
process. Consistent with the engagement of multiple cell death pathways during HSV-1 HF strain
infection, we next examined the role of caspase-1 in cytokine production and cell death. As
expected, Casp1−/− BMDMs displayed markedly reduced secretion of IL-1β and IL-18 following
infection, whereas substantial cell death persisted, indicating the involvement of caspase-1–
independent death pathways (Supplementary Fig. S4a, b). In contrast, loss of MyD88 or TRIF did
not affect cell death, indicating that Toll-like receptor signaling is dispensable in this context
(Supplementary Fig. S4c, d). Finally, deletion of cGAS, STING, or IRF1 significantly impaired
virus-induced cell death, phenocopying AIM2 deficiency, and supporting a model in which the
cGAS–STING–IRF1 axis licenses AIM2-dependent inflammasome activation and downstream
inflammatory cell death (Supplementary Fig. S4e, f, g). We also observed interactions between
ASC, AIM2, and caspase-1 following HF strain infection (Fig. 2f), indicating a distinct ability of this
strain to form the AIM2 inflammasome. Overall, these findings suggest that AIM2 inflammasome
activation and coordinated inflammatory cell death programs are crucial for host defense against
HSV-1 HF strain infection.

Strain-dependent contribution of VP22 to inflammasome activation during HSV-1 infection.
Previous studies have shown that the HSV-1 F strain viral protein VP22 modulates AIM2
inflammasome activation in BMDMs13, 14. To assess potential sequence variation among HSV-1
strains, we compared VP22 from the HF strain with those from the reference F and KOS strains
(Fig. 3a). This analysis identified five missense substitutions in HF VP22 (C29G, A49S, P51Q,
P135Q, and R141C).

Structural annotations indicate that VP22 contains a conserved core domain spanning amino
acids 169–254, defined based on sequence conservation and structural analyses across alpha
herpesvirus species32, 33, 34, 35. All identified HF-specific substitutions are located outside this
conserved core. However, regions outside the structured core are less well-characterized and
may include intrinsically disordered segments that mediate context-dependent interactions with
host factors. Accordingly, while VP22 exhibits strain-specific sequence variation, the functional
significance of these non-core substitutions, particularly in the context of inflammasome regulation,
cannot be inferred from sequence or structural information alone.

To examine the inhibitory effect of AIM2 inflammasome activation by VP22, we transfected VP22
from the F strain into WT and Aim2−/− BMDMs and subsequently infected them with the HF strain
of HSV-1 (Supplementary Fig. S5a). Infection with the HF strain of HSV-1 resulted in the cleavage
of caspase-1 (Supplementary Fig. S5b), release of inflammasome-driven cytokines IL-1β
(Supplementary Fig. S5c) and IL-18 (Supplementary Fig. S5d), and cell death (Fig. 3b, c) in an
AIM2-dependent manner, unaffected by the transfection of VP22 from the F strain. These findings
suggest that, unlike the inflammasome inhibitory effect of VP22 in the F strain of HSV-1, VP22 in
the HF strain does not play a role in AIM2 inflammasome activation.

To further evaluate the contribution of VP22 to AIM2 inflammasome regulation in the context of
viral infection, we next generated VP22-null mutant viruses in both the HF and F strain
backgrounds using CRISPR/Cas9-mediated genome editing (Supplementary Fig. S5e, f).
Successful deletion of VP22 was confirmed by immunoblot analysis, demonstrating complete loss
of VP22 protein expression in the mutant viruses (Supplementary Fig. S5g, h). Consistent with
the known role of VP22 in supporting optimal viral fitness, VP22-null viruses exhibited a modest
reduction in overall viral protein expression compared with their respective WT counterparts
(Supplementary Fig. S5g, h).

We first assessed the impact of VP22 deletion in the HF strain background. Infection of BMDMs
with either the WT HF or VP22-null HF virus resulted in comparable levels of caspase-1 cleavage
as well as similar secretion of IL-1β and IL-18 (Fig. 3d–f). These results indicate that deletion of
VP22 does not alter AIM2 inflammasome activation during HF strain infection, supporting the
conclusion that VP22 is dispensable for inflammasome regulation in this context. Together with
the results of the VP22 overexpression experiments, these findings suggest that the enhanced
AIM2 inflammasome activation observed during HF strain infection is not driven by VP22 and
instead points to other HF-specific genomic determinants. In contrast, a distinct outcome was
observed in the F strain background. While the WT F virus failed to induce detectable caspase-1
activation or inflammasome-dependent cytokine release, infection with the VP22-null F mutant
triggered robust caspase-1 cleavage and secretion of IL-1β and IL-18 (Fig. 3g–i). These findings
demonstrate that AIM2 inflammasome activation in the F strain is revealed upon VP22 deletion,
supporting its previously reported inhibitory role in inflammasome regulation in HSV-1 F and
related strains13, 14. Collectively, these results indicate a strain-dependent role of VP22 in
regulating AIM2 inflammasome activation during HSV-1 infection. While VP22 functions as a
negative regulator of inflammasome activation in the F strain, it is dispensable for inflammasome
control in the HF strain. Given the highly conserved nature of the VP22 core domain reported in
structural studies, these strain-specific differences are unlikely to reflect major intrinsic functional
divergence of VP22 itself. Rather, our findings support a model in which viral genomic features,
rather than viral proteins, play a dominant role in shaping AIM2-dependent innate immune
responses during HF strain infection.

HSV-1 strain HF containing poly(T) DNA sequences emerges as a crucial ligand for AIM2
inflammasome activation.
Given AIM2's known affinity for dsDNA and its role in forming an inflammasome against DNA
viruses6, 7, 8, 9, 10, 11, 12, we investigated the impact of viral DNA as a ligand for AIM2-mediated
inflammasome activation. Genomic DNA from HSV-1 strain HF induced ASC speck formation,
caspase-1 cleavage, release of inflammasome-driven cytokines IL-1β and IL-18, and cell death
in an AIM2-dependent manner, whereas genomic DNA from strain KOS did not (Fig. 4a–f). This
suggests that genomic DNA from HSV-1 strain HF functions as a ligand for AIM2 inflammasome
activation.

To identify specific DNA sequences serving as ligands for AIM2 inflammasome activation, we
designed primers targeting unique regions within the HSV-1 HF, F, and KOS sequences. Two
unique DNA sequences in the non-coding region of HSV-1, including, 5ʹ-
TTTTTTTTTTTTTTTTTTTTTTTTTTT-3’ (DQ889502.1) and 5ʹ-
CCCTGGGAGATGGGCCCACCGT-3’ (DQ889502.1) that were exclusive to the HF strain were
identified (Fig. 5a, Supplementary Fig. S6a). Among these, only 27-mer poly(T) containing viral
dsDNA sequences (#4 and #5) induced caspase-1 cleavage and cell death in an AIM2-dependent
manner (Fig. 5b, c, d, Supplementary Fig. S6b, c, d).

To further define the DNA sequence features driving AIM2 activation, we next examined whether
the observed HF-specific response could be attributed to general DNA composition rather than
sequence length or context. Consistent with previous reports showing that AIM2 can be activated
by various dsDNA ligands, including poly(dG:dC), in a GC-content–independent manner10, 36,
transfection of poly(dG:dC) duplexes into BMDMs induced caspase-1 cleavage and IL-1β
secretion (Supplementary Fig. S6e, f). However, comparative analysis of HSV-1 genomes
revealed that poly(G:C) tracts of comparable length (≤16 nucleotides) were present across HF, F,
and KOS strains, with no selective enrichment in the HF genome. Moreover, longer poly(G:C)
stretches were absent in all of the examined HSV-1 variants, making them unlikely candidates for
mediating HF-specific AIM2 inflammasome activation. In contrast, genomic analysis identified a
uniquely long poly(A:T) stretch, a 27-mer poly(T) sequence, present in the HF strain but absent
or substantially shorter in the F and KOS strains. This observation prompted us to directly assess
the length dependency of poly(T)-mediated AIM2 activation. Therefore, we synthesized double-
stranded poly(T) DNA of defined lengths (10, 30, and 90 nucleotides) and transfected them into
BMDMs. Robust caspase-1 cleavage and IL-1β release were observed exclusively in response
to the 90-mer poly(T), while shorter poly(T) fragments failed to induce significant inflammasome
activation (Supplementary Fig. S6g, h). These findings align with prior studies demonstrating that
efficient AIM2 inflammasome assembly requires dsDNA exceeding a critical length threshold.
Consistent with the ability of long poly(T) DNA to promote AIM2 inflammasome activation10, we
next examined whether poly(T)-containing DNA facilitates assembly of the AIM2 inflammasome
complex. Immunoprecipitation analysis revealed an enhanced association between ASC, AIM2,
and caspase-1 following stimulation with HF strain genomic DNA or the 27-mer poly(T)-containing
viral dsDNA sequence (#5) (Fig. 5e). These interactions were not observed under conditions in
which shorter poly(T) DNA failed to activate the inflammasome. Together, these data indicate that
the presence of poly(T)-rich viral DNA in the HF strain promotes physical assembly of the AIM2
inflammasome, providing a mechanistic basis for strain-specific inflammasome activation.

Poly(T) DNA length determines strain-specific AIM2 inflammasome activation and host
defense during HSV-1 infection.
To directly assess the role of HF-specific poly(T) DNA in inflammasome activation, we generated
a panel of recombinant HSV-1 HF viruses, including the WT HF strain (WT HF), a mutant lacking
the 14-mer poly(T) sequence in the UL25–UL26 intergenic region (HF ΔT14), and a revertant virus
(HF ΔT14-Rev) restoring the deleted poly(T) sequence (Supplementary Fig. S7a, b). All three
viruses exhibited comparable expression of representative viral proteins and showed no
differences in UL25 or UL26 transcript levels, indicating preserved viral gene expression and
intact neighboring loci (Supplementary Fig. S1c; Supplementary Fig. S7c, e). In primary BMDMs,
infection with WT HF or HF ΔT14-Rev robustly induced caspase-1 cleavage, secretion of IL-1β
and IL-18, and inflammatory cell death, whereas HF ΔT14 infection failed to elicit these responses
(Fig. 6a–e). Notably, all inflammasome-associated responses were completely abolished in
Aim2−/− BMDMs (Fig. 6a–e), confirming that poly(T)-mediated inflammasome activation by the HF
strain is strictly AIM2-dependent.

We next examined the impact of poly(T) deletion in vivo. WT mice infected with HF ΔT14 rapidly
succumbed to infection, whereas most WT HF–infected mice survived (Fig. 7a). Consistent with
this, viral titers were elevated and caspase-1 activation and IL-1β and IL-18 production in the
lungs were abolished in HF ΔT14-infected WT mice, phenocopying infection in Aim2−/− animals
(Fig. 7b–g). Restoration of the poly(T) sequence in HF ΔT14-Rev rescued survival, viral control,
and inflammasome activation to levels indistinguishable from WT HF infection (Fig. 7a–g). In
Aim2−/− mice, all three viruses caused uniformly lethal infection with no detectable inflammasome
activation, demonstrating that the protective effect of the poly(T) motif is entirely AIM2-dependent
in vivo.

To determine whether poly(T) DNA is sufficient to trigger AIM2 inflammasome activation, we
generated a recombinant HSV-1 F strain harboring a 27-mer poly(T) insertion in the UL25–UL26
intergenic region (F T27), along with a corresponding revertant virus (F T27-Rev) (Supplementary
Fig. S7b). In primary BMDMs, infection with F T27, but not WT F or F T27-Rev, induced robust
caspase-1 cleavage in an AIM2-dependent manner (Supplementary Fig. S8a). In vivo, WT mice
infected with F T27 exhibited significantly improved survival and enhanced caspase-1 activation
compared with those infected with WT F or F T27-Rev, whereas these responses were completely
abolished in Aim2−/− mice (Supplementary Fig. S8b–e). Viral protein expression and neighboring
gene transcription were comparable across all F strain variants, ruling out differences in viral
replication or fitness as confounding factors (Supplementary Fig. S1d; Supplementary Fig. S7d,
f).

Given the human specificity of HSV-1, we extended these findings to a human macrophage model.
In PMA-differentiated THP-1 cells, siRNA-mediated knockdown of AIM2 abolished inflammatory
cell death induced by WT HF, HF ΔT14-Rev, and F T27, while leaving responses to HF ΔT14, WT
F, and F T27-Rev unchanged (Supplementary Fig. S9a–f). Across all viral backgrounds and
genetic manipulations, the inflammasome phenotypes observed in murine BMDMs were faithfully
recapitulated in human macrophages, demonstrating conservation of poly(T)-mediated AIM2
activation across species.

Finally, to determine whether long poly(T) motifs represent viral genomic features present in both
clinical and laboratory isolates rather than solely culture-derived artifacts, we comprehensively
analyzed publicly available viral genome datasets. Among 492 complete HSV-1 genomes, both
14-mer and 23-mer poly(T) sequences were detected in multiple clinical isolates and laboratory
strains, with the 14-mer present in approximately 15% and the 23-mer in 3% of clinical samples
(Supplementary Fig. S10a, b). Moreover, long poly(T) tracts were identified across a broad range
of DNA and RNA viruses, including hepacivirus, monkeypox virus, SARS-CoV, adenovirus, and
herpes viruses (Supplementary Data 1, 2), indicating that these motifs are consistent with
naturally occurring variation and appear to be evolutionarily conserved and widespread.

Collectively, these loss-of-function, gain-of-function, rescue, and cross-species analyses provide
strong genetic and functional evidence that poly(T) DNA length is a key determinant of strain-
specific AIM2 inflammasome activation. Our findings establish viral poly(T) DNA as genome-
encoded and functionally relevant innate immune ligand that drives inflammatory cell death and
antiviral host defense during HSV-1 infection.

Methods
Mice
C57BL/6J mice (WT) were obtained from Hyochang Science, while Aim2−/− (Jackson Laboratory),
Nlrp3−/− (Cyagen), Nlrc4−/− (Cyagen), Casp1−/− (Jackson Laboratory) mice, all on a C57BL/6J
genetic background, were purchased from the indicated sources7, 12. All mice used in this study
were of the species Mus musculus. The mice were group-housed, with up to five mice per cage,
and were bred under standard pathogen-free conditions in the animal facility at the Ulsan National
Institute of Science and Technology (UNIST). The mice were maintained on a 12-h light/dark
cycle (lights on from 7 AM to 7 PM) at a controlled ambient temperature (22 ± 2°C) and relative
humidity (50 ± 10%), and were provided with standard chow. Both male and female mice were
included in this study. In vivo investigations utilized age- and sex-matched mice aged 6 to 8
weeks, while in vitro studies involved mice aged 6 to 12 weeks. Co-housed animals were chosen
for in vivo analyses. All experimental procedures were executed following protocols approved by
the Institutional Animal Care and Utilization Committee of UNIST [UNISTIACUC-23-22].

Cell culture
Primary bone marrow-derived macrophages (BMDMs) were cultured for 6 days in Iscove's
Modified Dulbecco's Medium (IMDM, Thermo Fisher Scientific, Cat. No. 12440061),
supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Cat. No. 16000044),
30% L929-conditioned medium, 1% non-essential amino acids (Thermo Fisher Scientific, Cat.
No. 11140-050), and 1% penicillin-streptomycin solution (Thermo Fisher Scientific, Cat. No.
15070-063). These BMDMs were seeded in 12-well plates at a density of 1 million cells/well and
incubated overnight before further use. L929 cells (ATCC, CCL-1) were procured and cultured in
IMDM supplemented with 10% FBS (Thermo Fisher Scientific, Cat. No. 16000044), 1% non-
essential amino acids, and 1% penicillin-streptomycin. Immortalized bone marrow-derived
macrophages (iBMDMs) were cultivated in Dulbecco’s Modified Eagle Medium (DMEM, Thermo
Fisher Scientific, Cat. No. 11995081) containing 10% FBS and 1% penicillin-streptomycin. THP-
1 cells were kindly provided by Atsushi Kawaguchi (University of Tsukuba) and cells were grown
in RPMI 1640 (Thermo Fisher Scientific, Cat. No. 11875119) with 10% FBS and differentiated into
macrophages in RPMI 1640 medium containing 20% FBS and 100 ng/ml PMA (SIGMA, Cat. No.
P1585-5MG) for 2 days. Vero cells (provided by Atsushi Kawaguchi (University of Tsukuba)),
293FT cells (Thermo Fisher Scientific, Cat. No. R70007), and SHaCaT cells (Cytion, Cat. No.
300493) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific,
Cat. No. 11995081) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific,
Cat. No. 16000044) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 15070-
063). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ and routinely
passaged to maintain exponential growth. All cell lines were tested and confirmed to be free of
mycoplasma contamination.

Generation of knockout clonal cells
WT and Gsdmd−/− iBMDMs were kindly provided by Professor Tae-Hyuk Kwon (UNIST). In
addition, Casp1−/−, Casp3−/−, Casp6−/−, Casp7−/−, Casp9−/−, Gsdme−/−, Mlkl−/−, Ripk3−/−,
Ripk3−/−Casp8−/−, Aim2−/−, and Irf1−/− iBMDMs had been generated previously, as described earlier
(Supplementary data 3)7, 12. For the present study, Sting−/−, cGas−/−, Myd88−/−, and Trif−/− iBMDMs
were newly generated using a CRISPR/Cas9-mediated genome-editing approach
(Supplementary data 3). The WT iBMDM cells underwent electroporation with 1 µg of Cas9-
encoding plasmid (addgene, #43945) and 1 µg of gRNA-expressing plasmid (addgene, #104174),
following the Amaxa 4D-Nucleofector program DS-136. For electroporation, a total of 4 × 105 cells
were employed. To create single-cell-derived knockout clones in iBMDMs, the transfected cells
were seeded at a low density (0.5 cells/well) in a 96-well plate. After a period of 10–14 days, each
well was scrutinized for the presence of viable single-cell-derived clones. These clones were
detached using trypsin, and genomic DNA extraction was performed using the DNeasy Blood &
Tissue Kit (Qiagen). For confirmation of the knockout of the target gene in the single-cell-derived
clones, genomic regions encompassing the Cas9 target site were subjected to amplification using
KAPA HiFi HotStart PCR polymerase (KK2502). The ensuing amplicons were further amplified
using TruSeq HT dual-index-containing primers to produce libraries for deep sequencing. The
sequencing of these libraries was accomplished using an Illumina iSeq equipped with a paired-
end sequencing system. The calculation of mutation frequencies was performed using the
MAUND program, accessible at https://github.com/ibs-cge/maund.

Gene silencing mediated by siRNA
siRNA against the AIM2 gene was purchased from Life Technologies. The nucleotide sequences
of the siRNAs used were as follows: 5ʹ-GACAUCUGGAGUUCAUAGCACCAUA-3ʹ and 5ʹ-
UAUGGUGCUAUGAACUCCAGAUGUC-3ʹ. For transfection, 15 pmol of siRNA was incubated
with 500 μl of Opti-MEM and 7.5 μl of Lipofectamine RNAi Max (Life Technologies) for 15 min at
room temperature. The mixture was transferred into 12-well plates at a density of 105 cells/well
for THP-1 macrophages. As a negative control, nonspecific scrambled siRNA was used.

Generation of Recombinant HSV-1 Viruses
Recombinant HSV-1 viruses were generated using a two-step CRISPR/Cas9-mediated
homologous recombination strategy37. To construct the HF ΔT14 mutant, in which 14 thymidines
were deleted from the 27T stretch located between the UL25 and UL26 genes of the HF strain,
293FT cells were co-transfected with a CRISPR/Cas9 plasmid expressing a guide RNA (gRNA#1)
targeting the UL25–UL26 intergenic region and a donor plasmid carrying a CMV promoter-driven
eGFP cassette flanked by homology arm sequences (HAS). To maintain expression of the
essential UL26 gene, an internal ribosome entry site (IRES) sequence was inserted between the
eGFP cassette and the UL26 start codon. Transfection was performed using jetOPTIMUS reagent
(Polyplus, 101000006), followed by treatment with 1 μM SCR7 (Sigma, SML1546) to inhibit non-
homologous end joining and 1 μg/mL puromycin (InvivoGen, ANT-PR-1) for selection. After 24 h,
cells were infected with WT HSV-1 HF at a multiplicity of infection (MOI) of 0.5. Viral progenies
were released by three freeze-thaw cycles, serially diluted, and used to infect Vero cells for plaque
purification. eGFP-positive plaques were identified using fluorescence microscopy, isolated, and
propagated to generate an intermediate recombinant virus containing an eGFP insertion between
UL25 and UL26. The final HF ΔT14 mutant was obtained by replacing the eGFP cassette with a
donor template carrying the ΔT14 sequence through a second round of CRISPR/Cas9
recombination using gRNA#2 targeting the CMV promoter. Recombinant viruses were plaque-
purified and verified by PCR and Sanger sequencing. CRISPR/Cas9 plasmids were constructed
by inserting synthesized oligonucleotides into pSpCas9(BB)-2A-Puro V2.0 (Addgene #62988),
and donor templates containing either the eGFP cassette or ΔT14 sequence flanked by homology
arm sequences were assembled using the AccuRapid Cloning Kit (Bioneer, K-7130). Additional
recombinant viruses, including HF ΔT14-Rev (revertant), F T27, and F T27-Rev, were generated
using the same procedure with strain-specific gRNAs and donor constructs specific to each strain.
HSV-1 VP22 knockout viruses (ΔVP22) were produced by the same strategy. Briefly, 293FT cells
were co-transfected with a CRISPR/Cas9 plasmid expressing a gRNA targeting the VP22 coding
region and a donor plasmid containing a CMV promoter-driven eGFP cassette flanked by
homology arms corresponding to sequences upstream and downstream of VP22. eGFP-positive
plaques were isolated using fluorescence microscopy and propagated in Vero cells. Deletion of
VP22 and insertion of the eGFP cassette were confirmed using PCR and immunoblotting with
anti-VP22 antibodies, and viral genome integrity was verified by Sanger sequencing.

Virus culture, infection, and cell stimulations
Human HSV-1 HF strain (ATCC; VR-260), HSV-1 F strain (ATCC; VR-733), and HSV-1 KOS
strain (ATCC; VR-1493) were purchased and propagated in Vero cells (gift from Dr. Atsushi
Kawaguchi, University of Tsukuba); the virus titer was measured using the plaque assay in Vero
cells. The HF ΔT14, HF ΔT14-Rev, F T27, F T27-Rev, and ΔVP22 mutant viruses were also
propagated in Vero cells. For HSV-1 (16 h; MOI 10) infection, primary BMDMs and iBMDMs were
cultured in DMEM high glucose (Gibco, 11995-065). For NLRP3 inflammasome activation, cells
were primed for 4 h with 500 ng/mL ultrapure LPS from Escherichia coli (0111:B4) (InvivoGen,
tlrl-3pelps) and then stimulated for 6 h with 1 μM nigericin (InvivoGen, tlrl-nig). For ligand-
mediated AIM2 inflammasome activation, 1 ng of poly(dA:dT) (InvivoGen, tlrl-patn), poly(dG:dC)
(InvivoGen, tlrl-pgcn), or poly(T) DNA of 10- , 30-, or 90-mer length was transfected into cells using
DOTAP (Roche, 11202375001) according to the manufacturer’s instructions, followed by
incubation for 6 h. For NLRC4 inflammasome activation, 10 ng of flagellin (Invivogen, tlrl-epstfla-
5) was transfected into cells using DOTAP (Roche, 11202375001) according to the
manufacturer’s instructions, followed by incubation for 6 h. In the context of VP22, F strain VP22
vector (pLV[Exp]-EGFP:T2A:Puro-EF1A>VP22) was purchased (Vector Builder, VB220920-
1383rfw) and BMDMs were transfected with the VP22 expression plasmid 12 h prior to viral
infection using DOTAP (Roche, 11202375001). Cells were then infected with HSV-1 and
harvested 16 h post-incubation. Genomic DNA from HSV-1 strain HF (ATCC, VR-260D) and
genomic DNA from HSV-1 strain KOS (ATCC, VR-1493DQ) were purchased and we transfected
1 ng of genomic DNA using DOTAP (Roche, 11202375001), following the manufacturer's
instructions. Cells were harvested at 16 h post-infection for immunoblotting, cytokine analysis,
ASC speck, and cell death quantification. For HSV-1 ligand screening, we transfected 1 ng of
indicated dsDNA using DOTAP (Roche, 11202375001), using the following dsDNA sets: 5ʹ-
TTTTTTTTTTTTTT-3ʹ, 5ʹ-TTTTTTTTTTTTTTTTTTTTTTTTTTT-3ʹ, 5ʹ-
CGTGAACGCAACGGCGGGAGCTGGGTTAACGTTTAGTTTAGCATTCGCTCTCGCCTTTCC
GCCCGCCCCCCGACCGTTGTGCCTTTTTTTTTTTTTT-3ʹ, 5ʹ-
GTTTAGTTTAGCATTCGCTCTCGCCTTTCCGCCCGCCCCCCGACCGTTGTGCCTTTTTTTTT
TTTTTTTTTTTTTTTTGTCCACCAA-3ʹ, 5ʹ-
GCCTTTTTTTTTTTTTTTTTTTTTTTTTTTGTCCACCAACGTCTCTGTGGGTGCGCGCATGGC
AGCCGATGCCCCGGGAG-3ʹ, 5ʹ-CCCTGGGAGATGGGCCCACCGT-3ʹ, 5ʹ-
CGTAACCATACCCAAACCGACTCTGGGGGTTGTTTGTGGGGSTCGGGACTATAGGATAAAC
AAACAGACCACCCCCGTACCTCCCCCTGGGAGATGGGCCCACCGTCCCAC-3ʹ, and 5ʹ-
CCCCGTACCTCCCCCTGGGAGATGGGCCCACCGTCCCACCCAAGGGTGCGGGTGGCTCA
TCGGCATCTGTGCGGTATTGGTTGTTACCCGC CCACTCGCGTTCGGACGT-3ʹ.

Cell death imaging and analysis
We employed an IncuCyte S3 imaging system (Sartorius). Cell death analysis was executed
following established methodologies7, 11, 12, 23, 28, 38, 39, 40, utilizing the same IncuCyte S3 imaging
system. The primary BMDMs, iBMDMs, or THP-1 were seeded into 12-well plates at a density of
106 cells/well for BMDMs, iBMDMs or 105 cells/well for THP-1 and subjected to stimulation. After
the stipulated incubation period, SYTOX Green (Thermo Fisher Scientific, S7020) was
administered following the manufacturer’s protocol. The ensuing images were subjected to
analysis using the software packaged with the IncuCyte imager.

Cytokine analysis
Cytokine levels were measured using IL-1β ELISA (Invitrogen, BMS6002TEN) and IL-18 ELISA
(R&D Systems, DY7625-05). For in vitro experiments, cell culture supernatants from infected
BMDMs were collected and centrifuged to remove cellular debris prior to cytokine quantification.
For in vivo experiments, bronchoalveolar lavage fluid (BALF) was harvested from infected mice.
All samples were analyzed according to the manufacturers’ instructions.

Immunoblotting analysis
Immunoblotting was conducted following established protocols7, 11, 12, 23, 28, 38, 40, 41, 42, 43, 44. To
assess caspase activity, BMDMs were lysed along with their supernatant using 50 μL of caspase
lysis buffer (comprising 1 × protease inhibitors, 1 × phosphatase inhibitors, 10% NP-40, and 25
mM DTT), followed by the addition of 100 μL of 4 × SDRS loading buffer. For signaling analysis,
BMDM supernatants were collected at specified time points, and after one wash with PBS, cells
were lysed with RIPA buffer. For all in vitro immunoblot analyses, BMDMs and iBMDMs were
seeded at 1 × 10⁶ cells per well, and lysates were prepared from the entire well. Equal numbers
of cells were processed under each condition, so additional protein concentration-based
normalization was not performed. Instead, equal volumes of lysates were loaded for SDS–PAGE,
and protein loading consistency was verified using internal control proteins, including β-actin or
GAPDH. For in vivo immunoblot analyses, approximately 100 mg of lung tissue from infected
mice was homogenized in cold RIPA buffer. Tissue lysates were clarified using centrifugation,
and protein concentrations were determined using a BCA protein assay. Lysates were then
adjusted to a final concentration of 2 mg/mL with the addition of RIPA buffer. Proteins were
resolved through electrophoresis on 8–15% polyacrylamide gels. After electrophoretic transfer
onto PVDF membranes (Millipore, IPVH00010), non-specific binding was blocked with 5%
skimmed milk. Subsequently, the membranes were incubated with the following primary
antibodies: anti-caspase-1 (AdipoGen, AG-20B-0042, 1:2000), anti-caspase-3 (CST, #9662,
1:2000), anti-cleaved caspase-3 (CST, #9661, 1:2000), anti-caspase-7 (CST, #9492, 1:2000),
anti-cleaved caspase-7 (CST, #9491, 1:2000), anti-caspase-8 (CST, #4927, 1:2000), anti-cleaved
caspase-8 (CST, #8592, 1:2000), anti-pMLKL (CST, #37333, 1:2000), anti-MLKL (Abgent,
AP14272b, 1:2000), anti-GSDMD (Abcam, ab209845, 1:2000), anti-β-actin (Proteintech, 66009-
1-IG, 1:5000), anti-ICP0 (Santa Cruz Biotechnology, sc‑53070, 1:1000), anti-ICP8 (Santa Cruz
Biotechnology, sc-53329, 1:1000), anti-gD (Santa Cruz Biotechnology, sc-21719, 1:1000), anti-
gC (Abcam, ab6509, 1:2000), anti-GAPDH (Santa Cruz Biotechnology, sc-365062, 1:3000), anti-
VP22 (kindly provided by Yasushi Kawaguchi, 1:2000), anti-CASP6 (CST, #9762, 1:2000), anti-
CASP9 (CST, #9504, 1:2000), anti-tRIPK3 (Prosci, #2283, 1:2000), anti-cGAS (CST, #31659,
1:2000), anti-STING (CST, #13647, 1:2000), anti-IRF1 (CST, #847S8, 1:2000), anti-ASC (Millipore,
#04-147), and anti-AIM2 (Abcam, ab119791, 1:2000). After washing, the membranes were
exposed to suitable horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted
1:5000; Jackson ImmunoResearch Laboratories, anti-rabbit [111-035-047], anti-mouse [315-035-
047]) for 1 h. Protein bands were visualized using Luminata Forte Western HRP Substrate
(Millipore, WBLUF0500), and the membranes were examined using Amersham ImageQuant 800
UV. The resulting images were analyzed utilizing ImageJ software (v1.53a). Uncropped
immunoblot images are provided in the Source Data file.

Immunofluorescence staining
Immunofluorescence staining was conducted as previously described7, 11, 12, 27, 43, 44. In brief, the
cells were fixed with 4% paraformaldehyde (PFA) for 10 min, permeabilized with PBS containing
0.5% Triton X-100 for 3 min, and subsequently incubated in PBS containing 1% skim milk for 1 h.
The coverslips were then incubated with anti-ASC (Millipore, 04-147) and anti-tubulin (CST, 2144)
antibodies. The secondary antibodies used were Alexa Fluor 488- conjugated anti-mouse IgG
(Life Technologies, A21202; 1:200) and Alexa Fluor 568-conjugated anti-rabbit IgG (Life
Technologies, A10042; 1:200). For visualization, DAPI mounting medium (P36931; Invitrogen,
Carlsbad, CA, USA) was used for counterstaining. The images were captured via a confocal laser
scanning microscope (LSM900; Carl Zeiss) equipped with a 63 × apochromatic objective.

Immunoprecipitation
Immunoprecipitation was performed following previously established protocols7, 11, 28, 44. Briefly, 1
× 10⁷ cells per condition were seeded and subjected to HSV-1 infection or stimulation as indicated.
Cells were then lysed in ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.4), 100 mM NaCl,
30 mM KCl, and 0.1% NP-40. Lysates were clarified by centrifugation at 16,000 × g for 10 min at
4°C to remove insoluble debris. Protein concentrations were quantified using a BCA protein
assay, and lysates were normalized to a final concentration of 1 mg/mL total protein. Equal
amounts of protein were used for each immunoprecipitation reaction. The normalized lysates
were incubated overnight at 4°C with either an IgG control antibody (CST, 3900S) or an anti-ASC
antibody (AdipoGen, AG-25B-006-C100), together with protein A Sepharose beads (GE
Healthcare, GE17-5280-01). Following extensive washing with the lysis buffer,
immunoprecipitated proteins were eluted using 0.1 mM glycine (pH 3.0).

RT-qPCR
HaCaT cells were infected with HSV-1 HF (WT, ΔT14, and ΔT14-Rev) or F (WT, T27, and T27-Rev)
strains at an MOI of 3. At 6 hpi, total RNA was extracted using the AccuPrep Universal RNA
Extraction Kit (Bioneer, K-3140). Complementary DNA (cDNA) was synthesized from the isolated
RNA using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems, 4387406). Quantitative
PCR was performed with the TOPreal SYBR Green qPCR High-ROX Premix (Enzynomics,
RT501M) to measure UL25 and UL26 transcript levels. Gene expression values were normalized
to ICP27 mRNA, which served as an internal control.

Analysis for HSV-1 Genomic Sequence and Protein Structure
Sequence alignment was performed using the Biopython package to compare the VP22 protein
sequences of HSV-1 strains HF (GenBank Accession No. DQ889502), F (GenBank Accession
No. GU734771), and KOS (GenBank Accession No. JQ673480). The Multiple Seq Alignment
module facilitated the identification of sequence variations, with a specific focus on missense
mutations exclusive to the HF strain. In addition to the above procedures, a comprehensive
search was conducted for the presence of specific sequences within the whole genomic sequence
of HSV-1. The three-dimensional structure of the VP22 protein was predicted using the SWISS-
MODEL server, which provided insights into the structural implications of the HF strain-specific
variants33. Subsequent modeling was performed to assess the effects of these variants on the
protein's overall structure.

Identification and Quantification of Poly(T) Tracts in HSV-1 Genomes
A total of 492 complete HSV-1 genome sequences, including reference strains, laboratory strains,
and clinical isolates, were obtained from the NCBI Virus database (Supplementary Data 1, 2).
Each genome assembly was computationally screened for homopolymeric thymidine [poly(T)]
tracts using custom Python scripts. poly(T) tracts of predefined lengths, poly(T)14 (≥14
consecutive thymidines) and poly(T)23 (≥23 consecutive thymidines), were identified across all
genomes. Each genome was annotated with binary indicators reflecting the presence or absence
of poly(T)14 and poly(T)23 motifs. Genomes were grouped according to strain designation,
including laboratory strains (H166, KOS63, RH2), clinical samples, and other annotated HSV-1
strains. The proportion of genomes containing poly(T)14 or poly(T)23 motifs was calculated for
each group as the percentage of positive samples relative to the total number of genomes within
the group. Percentage distributions were summarized and visualized using Python-based data
analysis workflows (pandas, matplotlib, and seaborn). Visualization parameters were
standardized to generate publication-quality figures. This analysis enabled comparative
assessment of poly(T) tract enrichment across HSV-1 strains and revealed that poly(T)23 motifs
were exclusively detected in H166, KOS63, RH2, and clinical isolates, whereas poly(T)14 motifs
were broadly distributed across the HSV-1 population.

In vivo infection
Age- and sex-matched cohorts of 6- to 8-week-old WT and Aim2–/– mice, housed together, were
utilized for the infection experiments. Mice were anesthetized with 250 mg/kg−1 avertin and then
infected intranasally with HSV-1 in 100 μL PBS containing approximately 5 × 105 PFU. Infected
mice were monitored over a period of 14 days for survival. Lung tissue collected at 5 days post-
infection were homogenized in 1 mL PBS for viral titers to be enumerated by plaque assays.

Statistical analysis
We performed data analysis using GraphPad Prism 10.0 software. The data are expressed as
the mean ± SEM or ± SE. We determined statistical significance using a two-tailed unpaired t-
test, a one-way ANOVA (Dunnett's multiple comparisons test), and two-way ANOVA
(Šídák's multiple comparisons test) with multiple comparisons for analyses involving multiple
groups. Survival curves were analyzed using the log-rank (Mantel–Cox) test. Statistical
significance was acknowledged for P values below 0.05 and denoted as *P < 0.05, **P < 0.01,
***P < 0.001, and ****P < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study focuses on the differences in AIM2 inflammasome activation among HSV-1 strains, revealing that the HF strain strongly stimulates the inflammasome, whereas the F and KOS strains show minimal activation. In this work, genomic analysis identified unique 14 bp poly(T) DNA sequences in the HF strain, located between the UL25 and UL26 genes, as critical for AIM2 inflammasome activation. Removing these sequences from the HF strain confirmed their importance, showing reduced inflammasome activation and compromised host defense. Transfection experiments with various lengths of poly(T) DNA further suggested that longer sequences are essential for AIM2 activation. These interesting findings not only clarify the role of poly(T) sequences in AIM2 inflammasome activation during HSV-1 infection but also suggest broader pathological implications of these sequences in other diseases. Understanding these interactions could lead to the development of novel therapeutic strategies against HSV-1 infections and other poly(T)-associated pathologies.\nThis work contributes to the current literature and brings new information to the field. Indeed, the work presented is original and provides important insights into a particular aspect of AIM2 inflammasome activation induced by different strains of HSV-1 both in vitro and in vivo. The authors have appropriately identified the work's hypothesis that the unique poly(T) DNA sequence of the HF strain interferes in AIM 2 inflammasome activation. The data are presented in a scientific manner, and all results are easy to interpret, making the scientific content easy to understand.\nInstead, the information regarding materials and methods needs to be increased to make the procedures used clearer.\nSpecific comments to the authors are as follow:\n1) Acronym should be explained as it’s the first time it appears in the text (AIM2/ASC/IL-1b/IL18/NLRP3/NLRC4/VP22 etc.).\n2) The manuscript would benefit from a thorough review of the terminology used, specifically the correct usage of HSV-1 and\ncaspase-1 to ensure accuracy and consistency throughout.\n3) In the first paragraph of the results, especially the in vivo part (Fig. 1h, 1i and 1j) I would suggest remarking the site of\ninfection in mice. Regarding the plaque assay I would suggest specifying the type of sample used (lung tissue) to facilitate\nthe reader's understanding.\nAlso in the first paragraph, I would suggest specifying which major inflammasome sensor you tested.\n4) I suggest clarifying any ambiguities in the methodology section, particularly regarding the techniques used for cytokine\nanalysis (what samples did you use? What procedure did you perform to prepare the samples?), protein quantification in\nWestern blots and immunoprecipitation.\n5) Provide additional discussion on the potential clinical implications of your findings and avenues for future research in this\nfield.\n6) I observed that for some western blots housekeeping is shown (Figures 1j, 6d) in others it is not (Figures 1c;3f and 4c).\nWhy?\n7) Also, why is actin housekeeping represented in Figure 2c for both caspase 1 and GSDMD?\nFurthermore, I suggest specifying the samples used for western blots in the supplementary materials images as well.\n8) Finally, in most of the western blots shown in Supplementary Materials, the marker is not present. Can you show it?", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_3": "AIM2 is a sensor of cytosolic double-stranded (ds)DNA that stimulates ASC- and caspase-1-dependent inflammasome\nactivation and subsequent IL-1β and IL-18 secretion and pyroptosis to combat various pathogens, including herpes simplex\nvirus 1 (HSV-1). It has been reported that, upon infection, HSV-1 dsDNA genomes activate AIM2-dependent\ninflammasomes, but the degree of activation during HSV-1 infection differs among HSV-1 strains. However, the detailed\nmechanism for the strain-specific differences remain uncertain. In this study, the authors addressed this question and found\nthat the variation in poly(T) sequence length in the intergenic region of the HSV-1 genome was responsible for the\ndifferences in AIM2 inflammasome activation. They demonstrated that the presence of a longer poly(T) sequence in the viral\ngenome robustly activated AIM2 inflammasomes in murine macrophages, and was linked to lower levels of pathogenicity\nand viral replication in mice in an AIM2-dpendent manner. This study presents some important and interesting findings\nregarding AIM2-dependent immune responses against HSV-1 infection. However, the experiments do not include critical\ncontrols and therefore, certain conclusions drawn by the authors lack sufficient evidence. More importantly, the mechanism\nby which the longer poly(T) sequence in the HSV-1 genome specifically activates AIM2 inflammasomes during viral\ninfection remains to be addressed.\nMajor Comments:\nComment 1: The cited literature (PMID: 22483801) reported that the HIN domain of AIM2, which is responsible for the\nbinding to ligand DNAs, interacts with DNAs containing 0%, 30%, 45%, and 90% GC content with similar efficiency.\nAdditionally, various types of DNAs, including poly(dG:dC), have been shown to efficiently activate the AIM2-dependent\ninflammasome (PMID: 20457908). Experimental data elucidating the molecular mechanism by which the longer poly(T)\nsequence specifically serves as a crucial ligand for AIM2 are required. Knowledge of this mechanism would contribute to a\nconceptual advance in the DNA recognition of sensor proteins in innate immunity.\nComment 2: To properly interpret the data presented in Fig. 1a–g, the efficiencies of HSV-1 HF, HSV-1 F, and HSV-1 KOS\ninfection in BMDMs need to be shown. The authors should analyze the expression levels of representative immediate-early,\nearly, and late HSV-1 proteins in these infected cells at various times after infection. Moreover, the authors should examine\nthe expression levels of VP22, which has been reported to inhibit AIM2 inflammasome activation, in infected cells at various\ntimes after infection.\nComment 3: Fig. 2 a–g. The effects of AIM2 knock-out on the expression of VP22 in BMDMs infected with each of the HSV-1\nstrains should be analyzed.\nComment 4: Fig. 3 c–e. Based on these results, the authors concluded that VP22 did not inhibit HSV-1-HF-induced AIM2\ninflammasome activation. However, the data do not include the expression levels of VP22 in transfected BMDMs, and\ntherefore, it is uncertain whether sufficient VP22 was expressed in these cells. The authors should analyze whether\ntransfection with the VP22 expression vector increased the levels of VP22 in the cells. Moreover, the authors should\nexamine the effects of the VP22-null mutation on AIM2 inflammasome activation (caspase-1 cleavage, IL-18, and IL-1β\nsecretion, and cell death) in BMDMs using VP22-null mutant HSV-1 HF together with VP22-null mutant HSV-1 F or KOS as\na control.\nComment 5: The authors should show whether the 23-mer poly(T) sequence is conserved in other laboratory strains and\nclinical isolates of HSV-1. Genetic modification (i.e., mutations associated with syncytial phenotype) sometimes artificially\noccurs during serial passages in cell cultures. How did the authors eliminate this possibility for the 23-mer poly(T) sequence\nin the HF strain?\nComment 6: Characterization of HFdelata14 is required. The authors should examine the effects of deltaT14 on viral\nreplication, infection efficiency, the expression levels of neighboring genes (UL20 and UL21), and VP22 expression levels in\nBMDMs. To eliminate the off-target effect(s) of CRISPR/Cas9-mediated genome editing, the authors should construct a\nrecombinant virus in which the mutation in HFdelta14 is repaired and test it together with wild-type and HFdelta14 viruses in\nthe experiments shown in Fig. 6 and Extended Data Fig. 4.\nComment 7: To further verify the authors’ conclusion that the length of the poly(T) sequence in the intergenic region of HSV-\n1 genome is critical for AIM2 inflammasome activation during HSV-1 infection, the authors should construct a recombinant\nvirus derived from HSV-1 F or a KOS strain harboring the 27-mer poly(T) sequence in the intergenic region, as well as its\nrepaired virus, and test them together with wild-type HSV-1 F or KOS in the experiments in Fig. 6 and Extended Data Fig. 4,\nafter characterization of the recombinant viruses, as described in Comment 6.\nComment 8: All analyses were conducted using mouse-derived cells. Since HSV-1 is a human pathogen, the authors\nshould perform representative experiments shown in Figs. 2–5 and Extended Data Fig. 4 using human-derived cells.\nMinor Comments\nComment 1: The ASC-citrine specks in Fig. 1a and Fig. 2a are unclear. Enlarged or higher-resolution images should be\nprovided to clearly delineate the ASC-citrine specks.\nComment 2: The authors should disclose detailed information about the VP22 expression plasmid, the gRNA sequences,\nand the names of the gRNA expression plasmids for the knockout iBMDMs, as well as sequence information for each gene\nafter genome editing.\nComment 3: The infection and transfection conditions in the experiments shown in Figs. 1 to 3 should be provided in more\ndetail. What multiplicity of infection was used? When were the samples harvested? When were the BMDMs transfected with\nthe VP22-expression plasmid?\nComment 4: In Extended Data Fig. 3a, the numbers (1)–(3) should be (6)–(8)?", "reviewer_4": "In the present work Oh et al study the inflammasome activation by Herpes Simplex Virus 1 strains in Mice and BMDMs and\nidentify a poly(T) sequence of the HF strain that specifically activate Aim2. Although it is well established that dsDNA induce\nAim2 activation, and that the Aim2 protein per se could have relatively broad specificity for different dsDNA, this study\nemphasis that in the cellular context the situation is very much more controlled and that (as far as I can see) the poly(T)\nsequence constitutes the first HSV1 sequence identified that can drive activation of innate immunity through Aim2. Due to\nthis, and when our understanding of the role of Aim2 in virus immunity is still quite fragmented, I feel this work could be well\nsuited for publication in Nature communication.\nOverall, the study comprise quite comprehensive data and although the methodology used (knockdowns, IP, etc) is often\nopen for multiple possible interpretations of what is going on in the cellular context, it appears as the data support most of\ntheir conclusions. However, I have few specific concerns that I feel should be addressed:\n- Several readout assays for Aim2 induction are used through the study, mainly Il1b/il18 release, Casp1 cleavage and cell\ndeath which in most cases appears to support a textbook view of Aim2 activation and signaling. However, the knockdown\nexperiments shown in Extended Data Fig 2 of proteins that could be involved downstream of Aim2 activation is poorly\nfollowed up and the apparent observation that cell death is not dependent on Casp1 or GSDMD/E is intriguing and would\ncontradict the textbook view. In a HSV1 infection (and the transfection of full virus DNA) it is quite possible that multiple\npathways are activated and, although it is clear that Aim2 signaling is one of these, there might be other pathways\ncontributing to e.g. to cell death and Il1b/Il18 release in parallel (Yes, the Aim2 KD support that Aim2 is a key player, but\nthese are KD clones with all the possible caveats, where compensatory and complex distortions of immune signaling might\nhave been induced due by KD of Aim2). The Casp1 or GSDMD KD data is confusing and it is also possible that these\nknockdown experiments have not worked well/are not informative (where are the WBs showing that these proteins are\nindeed knocked down?). I feel the authors should add some further follow-up experiment on at least the Casp1 knockdown\nto show whether IL1b/Il18 release is attenuated in the BMDMs knockdown as expected. Overall, the mechanistic\ndiscussions are very sparse and the work would benefit from the authors further develop there point on e.g. RIPK3\ndependence in the discussion and potentially also add clarifying experiments. Can it be excluded that there is a significant\nToll receptor effect contributing to Il1b/Il18 release or apoptosis? Etc\n- The section addressing a potential role of VP22 is an inhibitor of Aim2 is not very crisp and the experiments might\neventually not be informative. The initial paragraph contains a somewhat superficial discussion of structural regions and\nwhat could be functionally significant. In the end of a paragraph, they say “Variants outside of this core domain are thus not\nexpected to significantly impact VP22 functions and are consequently categorized as functionally neutral”. Although e.g.\nAints et al addresses other types of functions, I cannot really find the data excluding that other regions could have additional\nfunctions, e.g. in the context of Aim2 inhibition. Although these regions might be intrinsically disordered, they could make\nimportant interactions. This paragraph should be rewritten, e.g. to instead just describe what is known about the structure (if\nthe authors do not know about more crisp data excluding functional significance of specific regions).\n- It is concluded from the VP22 over-expression experiment that there is no differential effect of VP22 for the different strains.\nHowever, there is no positive control experiment showing that the VP22 overexpression in this system can inhibit Aim2.\nTherefore, I would like to see a control experiment demonstrating that VP22 overexpression can indeed inhibit Aim2\nactivation, e.g. with a highly potent dsDNA for Aim2 activation. Otherwise, there is no use addressing differential effects.\n- In the IP figures (e.g. Fig2 F), in the Input chart, there is no Aim2 protein present for the WT media lane. This suggests that\nthe virus first induce Aim2, which is subsequently activated by viral dsDNA (?). How is Aim2 induced in this context? It\nwould be particuliarly interesting to shed light on how this is accomplished for HF seq#5 when this is a requirement for Aim2\nactivation.\n- I feel the Discussion section is quite thin, and in addition to above points on the lack of discussion of mechanistic\nimplications, it should be possible to further address the implications of the poly(T) sequence for virus immune response. Are\nthere other Herpesviruses, other viruses, containing similar sequences. This sequence is relative short in relationship to\nsome other Aim2 inducing sequences, etc"}, "author_response": "We greatly appreciate the time and expertise the four reviewers have contributed to the\npeer review process. We are grateful for the constructive feedback which has substantially\nimproved our manuscript.\nIn the revised version of our manuscript, we have carefully considered and thoroughly\naddressed all constructive comments raised by the reviewers. We have worked extremely\nhard to generate a tremendous amount of new insightful data and to improve the quality\nof our manuscript and strengthen our conclusions following the expert suggestions of the\nreviewers. We have listed below new data that we have generated for the revised\nmanuscript. These new results show that:\n• Poly(dG:dC) transfection in BMDMs induces inflammasome activation, evidenced by\ncaspase-1 cleavage and IL-1β secretion, validating previous evidence of AIM2\nresponsiveness to GC-rich DNA.\n• All HSV-1 genomes analyzed (HF, F, KOS) contain poly(G:C) tracts ≤16 bp, a length\ncommonly found in HSV-1 strains and insufficient to explain strain-specific AIM2\nactivation, particularly in HF.\n• Only 90-mer poly(T) DNA, but not 10-mer or 30-mer, induces robust caspase-1\nactivation and IL-1β release, establishing a length-dependent mechanism of AIM2\nactivation likely mediated by higher-order DNA structures.\n• All three HSV-1 strains (HF, F, KOS) can successfully infect HaCaT and BMDM cells,\nwith comparable expression of viral proteins (ICP0, ICP8, gD, gC, and VP22), ruling out\ninfection efficiency as a variable in AIM2 activation.\n• Successful VP22 overexpression was directly validated by immunoblotting, excluding\ninsufficient protein expression as a reason for the lack of inhibitory activity.\n• A VP22-null mutant virus in the HF background (HF ΔVP22) shows no difference in\ninflammasome activation compared with WT HF, as measured by caspase-1 cleavage\nand IL-1β/IL-18 secretion, demonstrating that VP22 is dispensable in this context.\n• In the HSV-1 F strain background, deletion of VP22 (F ΔVP22), but not WT F infection,\nrestores robust AIM2-dependent inflammasome activation, including caspase-1\ncleavage and IL-1β/IL-18 secretion, demonstrating that VP22 acts as a strain-specific\ninhibitor of AIM2 in F but not in HF, where poly(T)-mediated activation predominates.\n• Analysis of 492 public HSV-1 genomes revealed that the 23-mer poly(T) sequence is\npresent in both clinical and laboratory isolates, including H166, KOS63, and multiple\npatient-derived strains, confirming it is not a cell-culture artifact.\n• The 14-mer poly(T) sequence is found in 15% of clinical HSV-1 genomes, and the 23-\nmer in 2.5%, supporting poly(T) length variation as a natural polymorphism among\nHSV-1 strains.\n• Cross-species viral genome analysis shows that 23-mer poly(T) sequences are found\nin multiple DNA and RNA viruses, including Monkeypox virus, adenovirus, Hepacivirus,\nand SARS-CoV, indicating conserved innate immune relevance.\n1\n• WT HF, HF ΔT , and HF ΔT -Rev viruses exhibit similar expression of viral proteins\n14 14\n(ICP0, ICP8, gD, gC, VP22) and neighboring genes UL25/UL26, confirming the poly(T)\ndeletion does not affect viral fitness or local transcriptional context.\n• Reversion of ΔT mutation (HF ΔT -Rev) restores inflammasome activation and host\n14 14\nprotection in vivo, including survival, lung viral titers, caspase-1 activation, and\ncytokine responses, all lost in the ΔT mutant and absent in AIM2-deficient mice.\n14\n• In vitro, HF ΔT -Rev infection restores caspase-1 cleavage, IL-1β/IL-18 secretion, and\n14\npyroptosis in BMDMs, mirroring WT HF responses and confirming strict AIM2\ndependence for poly(T)-mediated inflammasome activation.\n• F strain harboring a 27-mer poly(T) insertion (F T ) induces inflammasome activation\n27\nand improves survival in WT mice, but not in AIM2 KO mice, confirming sufficiency and\nAIM2 dependence of the poly(T)–mediated response.\n• F T infection activates caspase-1 and cytokine release in WT BMDMs, but not in AIM2\n27\n• In human THP-1 macrophages, AIM2 knockdown abolishes cell death in response to\nHF and F T infections, validating conservation of poly(T)-AIM2 sensing in human\n27\ninnate immunity.\n• VP22 does not suppress AIM2-dependent responses in human THP-1 cells, consistent\nwith murine data, and deletion of poly(T) in HF abrogates AIM2-dependent cell death,\nrescued by the revertant.\n• Insertion of poly(T) in F (F T ) enables cell death in human cells, which is completely\n27\nlost upon AIM2 silencing, confirming poly(T) sufficiency and AIM2 specificity.\n• MYD88 KO and TRIF KO iBMDMs show no difference in HSV-1 HF-induced cell death,\nexcluding significant contribution from TLR pathways in the observed inflammasome\nresponse.\n• cGAS, STING, and IRF1 knockout cells exhibit impaired AIM2-dependent cell death,\nsupporting a cGAS–STING–IRF1–AIM2 axis in viral sensing.\nWe would also like to highlight that our original manuscript has already demonstrated that:\n• HSV-1 induced inflammasome activation is dependent on the HF strain of HSV-1.\n• AIM2 is identified as an inflammasome sensor during HSV-1 infection.\n• Inflammasome sensors, including NLRP3 and NLRC4, do not play a role in activation\nor cell death during infection.\n• AIM2-dependent cell death during HSV-1 infection is characterized by pyroptotic\nmarkers (CASP1, GSDMD), apoptotic markers (CASP8, CASP3, CASP7), and\nnecroptotic markers (MLKL), indicating inflammatory cell death.\n• Deletion of cell death executioner molecules, including RIPK3 and CASP8, but not\nCASP1, CASP3, CASP6, CASP7, CASP9, GSDMD, GSDME, and MLKL, is sufficient to\nprotect cells from death induced by HSV-1 infection.\n• Previously known AIM2 inflammasome inhibitors such as the viral protein VP22 do not\nplay a role in inflammasome activation or cell death during HSV-1 strain HF infection.\n2\n• Viral DNA of HSV-1 strain HF, but not viral DNA of strain KOS, is sufficient to induce\ninflammasome activation and cell death.\n• The unique regions of the 14-mer poly(T) DNA sequence of the HF strain are identified\nwithin the HSV-1 HF, F, and KOS viral DNA sequences.\n• The mutant HSV-1 strain HF, with the 14-mer poly(T) DNA sequence deleted (HF ΔT ),\n14\nis insufficient to induce inflammasome activation and cell death.\n• The 14-mer poly(T) DNA sequence is critical for AIM2 sensing and host defense against\nHSV-1 infection.\n• WT mice succumb to HF ΔT HSV-1 infection within 11 days, while 80% of mice survive\n14\nagainst WT HSV-1.\n• Mice lacking AIM2 succumb to both WT and mutant HSV-1 infection within 9 and 10\ndays, respectively.\nOverall, our study uncovers the HSV-1 ligand for AIM2 inflammasome sensor but also\nestablishes the molecular basis of how this ligand can trigger the AIM2 inflammasome for\nhost defense. We firmly believe that the large amount of new data added responds to all of\nthe reviewers’ expert comments thoroughly and greatly improves the quality of our\nmanuscript. We have provided substantial new mechanistic data and multiple lines of\nevidence pointing toward the prominent role of a critical interaction between AIM2 and the\nPoly(T) DNA sequence. These findings represent a new paradigm in understanding the\nfunctions of these molecules in inflammasome and innate immunity, further strengthening\nthe central conclusions of our manuscript.\nThis study focuses on the differences in AIM2 inflammasome activation among HSV-1 strains,\nrevealing that the HF strain strongly stimulates the inflammasome, whereas the F and KOS strains\nshow minimal activation. In this work, genomic analysis identified unique 14 bp poly(T) DNA\nsequences in the HF strain, located between the UL25 and UL26 genes, as critical for AIM2\ninflammasome activation. Removing these sequences from the HF strain confirmed their\nimportance, showing reduced inflammasome activation and compromised host defense.\nTransfection experiments with various lengths of poly(T) DNA further suggested that longer\nsequences are essential for AIM2 activation. These interesting findings not only clarify the role of\npoly(T) sequences in AIM2 inflammasome activation during HSV-1 infection but also suggest\nbroader pathological implications of these sequences in other diseases. Understanding these\ninteractions could lead to the development of novel therapeutic strategies against HSV-1\ninfections and other poly(T)-associated pathologies.\nThis work contributes to the current literature and brings new information to the field. Indeed, the\nwork presented is original and provides important insights into a particular aspect of AIM2\ninflammasome activation induced by different strains of HSV-1 both in vitro and in vivo. The\nauthors have appropriately identified the work's hypothesis that the unique poly(T) DNA sequence\nof the HF strain interferes in AIM 2 inflammasome activation. The data are presented in a scientific\nmanner, and all results are easy to interpret, making the scientific content easy to understand.\nInstead, the information regarding materials and methods needs to be increased to make the\nprocedures used clearer.\nWe sincerely thank you for your thoughtful and generous evaluation of our work. We are\nespecially grateful for the recognition that our study “brings new information to the field”\nand “provides important insights into a particular aspect of AIM2 inflammasome\nactivation.” We also appreciate your positive remarks on the clarity and scientific\npresentation of our data.\nWe have carefully addressed your comment regarding the need for more detailed\nmethodological information. In response, we have significantly expanded the Materials\nand Methods section to provide clearer descriptions of experimental procedures, including\ninfection conditions, transfection timing, and gene editing protocols. Additional details\nhave also been included in the figure legends and supplementary materials to enhance\nreproducibility.\nWe are truly grateful for your constructive feedback, which has helped us improve both\nthe scientific rigor and clarity of our manuscript.\nSpecific comments to the authors are as follow:\n1) Acronym should be explained as it’s the first time it appears in the text (AIM2/ASC/IL-\n1b/IL18/NLRP3/NLRC4/VP22 etc.).\nis introduced as “absent in melanoma 2 (AIM2),” and similarly ASC, IL-1β, IL-18, NLRP3,\nNLRC4, and VP22 are all spelled out at their initial appearance. This ensures that readers\nunderstand each term when it is first mentioned.\n2) The manuscript would benefit from a thorough review of the terminology used, specifically the\ncorrect usage of HSV-1 and caspase-1 to ensure accuracy and consistency throughout.\nWe thoroughly reviewed the manuscript for consistency in terminology. We have corrected\ninstances of inconsistent notation – for instance, we have now uniformly used “HSV-1”\n(with a hyphen) and “caspase-1” throughout the text. All virus strain names and\ngene/protein names are presented with correct and consistent formatting.\n3) In the first paragraph of the results, especially the in vivo part (Fig. 1h, 1i and 1j) I would suggest\nremarking the site of infection in mice. Regarding the plaque assay I would suggest specifying\nthe type of sample used (lung tissue) to facilitate the reader's understanding.\nAlso in the first paragraph, I would suggest specifying which major inflammasome sensor you\ntested.\nInfection Route (In vivo Experiments): We have clarified the site of infection in our in vivo\nexperiments. The revised Results section now explicitly states that mice were intranasally\ninfected with HSV-1 (5×10^5 PFU in 100 µL, as described in the Methods). This additional\ndetail makes the infection route clear for the experiments associated with Figure 1h–j.\nPlaque Assay Sample: We have specified the sample source for the plaque assays in the\ntext. In the Results, we now note that the viral titers (Figure 1h) were determined using\nlung tissue homogenates collected from infected mice. Stating that lung tissue was used\nfor the plaque assay helps the reader understand the origin of the samples for viral load\nmeasurements.\nInflammasome Sensor Tested: We have identified the major inflammasome sensor\nexamined in our study to avoid ambiguity. In the revised first paragraph of the Results, we\nclearly state that we focused on the AIM2 inflammasome pathway. For example, we\nmention that experiments were performed in AIM2-knockout macrophages to confirm\nAIM2’s role in HSV-1–induced inflammasome activation (shown in Fig. 2a, b). This\nclarification makes it explicit that AIM2 is the primary sensor being tested in our\ncomparisons.\n4) I suggest clarifying any ambiguities in the methodology section, particularly regarding the\ntechniques used for cytokine analysis (what samples did you use? What procedure did you\nperform to prepare the samples?), protein quantification in Western blots and immunoprecipitation.\nAdditional details have been added to the Methods to clarify how cytokine levels were\nmeasured. We now specify the sources of samples and preparation steps for IL-1β and IL-\n5\n18 assays. For instance, the Methods section now explains that cell culture supernatants\nfrom infected bone-marrow-derived macrophages were collected and centrifuged to\nremove debris before IL-1β and IL-18 quantification. In the case of in vivo experiments, we\nnow note that bronchoalveolar lavage fluid (BALF) from infected mice was harvested for\ncytokine measurements. These samples were then analyzed by ELISA following the\nmanufacturer’s protocols. These clarifications ensure that the procedures for cytokine\nanalysis are transparent and reproducible.\nAdditional details have been added to the Methods to clarify protein quantification\nprocedures for Western blotting and immunoprecipitation. For in vitro immunoblot\nanalyses, cells were seeded at fixed densities (1 × 10⁶ cells per well for BMDMs and\niBMDMs, and 1 × 10⁵ cells per well for THP-1 cells), and lysates were prepared from the\nentire well. Because equal numbers of cells were processed under each condition,\nadditional protein concentration–based normalization was not performed. Instead, equal\nvolumes of lysates were loaded for SDS–PAGE, and protein loading consistency was\nverified using internal control proteins, including β-actin or GAPDH.\nFor in vivo immunoblot analyses, approximately 100 mg of lung tissue was harvested from\ninfected mice and homogenized in cold RIPA buffer. Tissue lysates were clarified by\ncentrifugation, and protein concentrations were determined using a BCA protein assay.\nLysates were then adjusted to a final concentration of 2 mg/mL with the addition of RIPA\nbuffer, and equal amounts of total protein were loaded for SDS–PAGE followed by\nimmunoblotting with the indicated antibodies.\nFor immunoprecipitation experiments, cells were seeded at a density of 1 × 10⁷ cells per\ncondition and infected as indicated. After infection, cells were lysed in ice-cold lysis buffer,\nand lysates were clarified by centrifugation to remove insoluble debris. Protein\nconcentrations were quantified using a BCA protein assay, and lysates were subsequently\nnormalized to a final concentration of 1 mg/mL total protein. Equal amounts of total protein\nwere used for each immunoprecipitation reaction, followed by incubation with the\nindicated antibodies and protein A/G beads under identical conditions. These procedures\nensured equivalent protein input across samples and enabled reliable comparison of\nimmunoprecipitated complexes. Together, these revisions clarify the procedures for\ncytokine analysis, protein quantification in Western blotting, and immunoprecipitation,\nensuring that all experimental methods are described in a transparent and consistent\nmanner so that they are reproducible.\n5) Provide additional discussion on the potential clinical implications of your findings and avenues\nfor future research in this field.\nThank you for this insightful suggestion regarding the clinical implications of our findings\nand directions for future research. In the revised Discussion section, we have expanded\nour interpretation of the data to explicitly address these points.\na dominant AIM2 ligand. We highlight that selective modulation of DNA–inflammasome\ninteractions, such as targeting poly(T)-rich genomic regions or downstream AIM2-\ndependent signaling pathways, may represent a potential strategy to attenuate excessive\ninflammasome activation and immunopathology during severe HSV-1 infection, while\npreserving antiviral host defense. Conversely, we also outline the possibility that\nenhancing AIM2 responsiveness to such DNA motifs could be explored as a means to\nbolster protective immunity in immunocompromised cases.\nIn addition, we have expanded the Discussion to emphasize broader implications beyond\nHSV-1. We note that long poly(T) tracts are present not only in laboratory-adapted strains\nbut also in multiple clinical HSV-1 isolates and across diverse viral families. This\nobservation raises the possibility that similar DNA motifs may function as innate immune\ntriggers in other viral or inflammatory contexts. Accordingly, we outline future research\ndirections, including systematic profiling of poly(T)-rich genomic regions across viral\ngenomes, investigation of how poly(T) length polymorphisms correlate with disease\nseverity and immune outcomes in vivo, and exploration of AIM2-dependent sensing\nmechanisms in related viral infections.\nTogether, these additions broaden the clinical and conceptual context of our findings and\nclearly define avenues for future investigation, in accordance with your request.\n6) I observed that for some western blots housekeeping is shown (Figures 1j, 6d) in others it is\nnot (Figures 1c;3f and 4c). Why?\nThank you for pointing this out. In our revised submission, we have ensured consistency\nin our presentation of housekeeping controls. For all Western blots presented in the main\nand supplementary figures, β-actin or GAPDH was used as the housekeeping control and\nwas consistently run and imaged. In cases where the original figure panels did not include\nthe actin blots, we have now either provided the corresponding actin panels or explicitly\nclarified this in the figure legends and Supplementary Figures. We sincerely apologize for\nthe oversight in initial figure assembly and have addressed it to improve transparency.\n7) Also, why is actin housekeeping represented in Figure 2c for both caspase 1 and GSDMD?\nFurthermore, I suggest specifying the samples used for western blots in the supplementary\nmaterials images as well.\nThank you for this valuable comment regarding the use of β-actin as a housekeeping\ncontrol in Figure 2c. In our study, different lysis buffers were used to extract caspase-1\nand GSDMD proteins owing to their biochemical properties. Specifically, caspase-1 was\nextracted using CASP lysis buffer optimized for caspase activity and stability, whereas\nGSDMD was extracted using RIPA buffer, which is suitable for solubilizing membrane-\nassociated proteins. As these two proteins were probed from separately prepared lysates,\nwe appropriately used independent β-actin blots as internal loading controls for each\n7\nprotein in Figure 2c. In the revised version of the manuscript, we have clarified this point\nin the figure legend to avoid confusion and to ensure transparency in data interpretation.\nFurthermore, in response to your suggestion, we have now specified the sample types\nused for all Western blot experiments in the supplementary materials (e.g., whole-cell\nlysates from BMDMs, lung homogenates from infected mice, etc.). To further enhance\nclarity and traceability of sample identities (such as WT versus AIM2-deficient samples),\nwe have additionally provided the corresponding uncropped original Western blot images\nalongside the cropped panels in the Supplementary Materials. These uncropped blots\ninclude full lane information and labeling, allowing unambiguous identification of each\nsample used in the presented figures. Together, these revisions improve clarity,\nreproducibility, and methodological rigor of the Western blot data.\n8) Finally, in most of the western blots shown in Supplementary Materials, the marker is not\npresent. Can you show it?\nWe completely understand your concern and fully agree that molecular weight markers\nare valuable for verifying protein band sizes in Western blotting. Unfortunately, owing to\nlimitations of our current imaging system (Amersham ImageQuant 800 UV), which relies\non chemiluminescence detection without integrated marker-lane annotation functionality,\nmolecular weight markers were not captured in the final blot images. Although the system\noffers high sensitivity for detecting protein signals, it does not allow co-visualization of\nmarkers and targets in a single image without additional processing steps, which are\ntechnically challenging under our current experimental setup.\nNevertheless, we consistently used pre-stained molecular weight markers during\nelectrophoresis for every blot. Band sizes were confirmed according to expected\nmolecular weights and verified using specific antibodies, with knockout cell lines serving\nas negative controls to confirm antibody specificity and band identity. While the markers\nare not visible in the images, we ensured that size determination and validation were\nrigorously performed.\nWe hope this explanation clarifies the technical constraints and reassures the reviewer of\nthe integrity and interpretability of the presented Western blot data.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nWe appreciate your participation in the co-review process and the Nature Communications\ninitiative to support Early Career Researchers. No further comments were provided in this\nsection, so we thank you for your support.\nAIM2 is a sensor of cytosolic double-stranded (ds)DNA that stimulates ASC- and caspase-1-\ndependent inflammasome activation and subsequent IL-1β and IL-18 secretion and pyroptosis to\ncombat various pathogens, including herpes simplex virus 1 (HSV-1). It has been reported that,\nupon infection, HSV-1 dsDNA genomes activate AIM2-dependent inflammasomes, but the\ndegree of activation during HSV-1 infection differs among HSV-1 strains. However, the detailed\nmechanism for the strain-specific differences remain uncertain. In this study, the authors\naddressed this question and found that the variation in poly(T) sequence length in the intergenic\nregion of the HSV-1 genome was responsible for the differences in AIM2 inflammasome activation.\nThey demonstrated that the presence of a longer poly(T) sequence in the viral genome robustly\nactivated AIM2 inflammasomes in murine macrophages, and was linked to lower levels of\npathogenicity and viral replication in mice in an AIM2-dpendent manner. This study presents some\nimportant and interesting findings regarding AIM2-dependent immune responses against HSV-1\ninfection. However, the experiments do not include critical controls and therefore, certain\nconclusions drawn by the authors lack sufficient evidence. More importantly, the mechanism by\nwhich the longer poly(T) sequence in the HSV-1 genome specifically activates AIM2\ninflammasomes during viral infection remains to be addressed.\nWe sincerely thank the you for your thoughtful and critical evaluation of our manuscript.\nWe greatly appreciate your recognition that our study presents “important and interesting\nfindings regarding AIM2-dependent immune responses against HSV-1 infection” and that\nit provides new insight into the strain-specific differences in inflammasome activation\ndriven by HSV-1 genomic variation.\nWe fully agree that a more detailed mechanistic understanding of how longer poly(T)\nsequences specifically activate AIM2 inflammasomes is of great importance, and we have\nmade efforts to address this in the revised manuscript. Additionally, we have conducted\nfurther experiments to include missing controls and strengthen the evidence for our\nconclusions, as suggested.\nYour constructive comments have helped us significantly refine the depth, clarity, and\nrigor of our study, and we are grateful for your valuable input.\nMajor Comments:\nComment 1: The cited literature (PMID: 22483801) reported that the HIN domain of AIM2, which is responsible for the\nbinding to ligand DNAs, interacts with DNAs containing 0%, 30%, 45%, and 90% GC content with similar efficiency.\nAdditionally, various types of DNAs, including poly(dG:dC), have been shown to efficiently activate the AIM2-dependent\ninflammasome (PMID: 20457908). Experimental data elucidating the molecular mechanism by which the longer poly(T)\nsequence specifically serves as a crucial ligand for AIM2 are required. Knowledge of this mechanism would\ncontribute to a conceptual advance in the DNA recognition of sensor proteins in innate immunity.\nAIM2 activation and the relevance of our poly(T) findings in light of prior reports showing\nGC-content-independent binding of the AIM2 HIN domain (PMID: 22483801) and\npoly(dG:dC)-mediated inflammasome activation (PMID: 20457908).\nTo address this, we performed additional comparative experiments. We transfected\nBMDMs with poly(dG:dC) duplexes and confirmed that they indeed induce caspase-1\ncleavage and IL-1β secretion, consistent with previous findings (New Data, Extended Data\nFigure 6e, 6f [shown below]). However, when comparing the HSV-1 HF strain to the F and\nKOS strains, we noted that all three genomes contained poly(G:C) tracts of 16 nucleotides\nor fewer, which are commonly found across HSV-1 strains and are not enriched in HF.\nImportantly, poly(G:C) sequences longer than 16 nucleotides were not detected in any\nvariant, making them unlikely candidates for explaining HF-specific AIM2 activation.\nIn contrast, we identified a unique 27-mer poly(A:T) sequence, especially poly(T), in the HF\nstrain, which was absent or significantly shorter in the F and KOS strains. This sequence\nvariation led us to hypothesize that poly(A:T), particularly poly(T), contributes to the strain-\nspecific AIM2 activation observed.\nTo test this, we synthesized double-stranded poly(T) DNA of 10-, 30-, and 90-nucleotide\nlengths and transfected them into BMDMs. Robust caspase-1 activation and IL-1β release\nwere observed only with the 90-mer poly(T), whereas the 10-mer and 30-mer failed to\ninduce significant caspase-1 cleavage or IL-1β release (New Data, Extended Data Figure\n6g, 6h [shown below]). This finding is consistent with a previous study (PMID: 22483801),\nwhich demonstrated that double-stranded DNA of at least 80 base pairs is required for\nefficient AIM2 inflammasome activation. These results suggest that poly(T)-mediated AIM2\nactivation is highly length-dependent, likely reflecting the requirement for higher-order\nDNA structures that facilitate AIM2 oligomerization and inflammasome assembly.\n11\n1PSAC\nTW\n–/–2miA\nP45\nP20\nActin\nTW\n–/–2miA\nTW\n–/–2miA\nA: d\nT)\nG: d\nC)\nM\nock\nP\noly(d\nP\noly( d\n200\n160\n120\n80\n–/–2miA\nTW\n–/–2miA\n40\n)lm/gp(\nβ1-LI\n****\n****\nA: d\nT)\nG: d\nC)\nP\noly(d\nP\noly(\nd\nCollectively, these findings support the idea that while AIM2 can indeed respond to various\nDNA ligands in vitro, in the cellular context of HSV-1 infection, the presence of a uniquely\nlong poly(T) stretch in the HF genome serves as a potent and physiologically relevant AIM2\nligand, capable of overcoming viral immune evasion mechanisms and triggering a\nstronger inflammasome response. We have included these new data in the revised\nResults and believe they help clarify the specificity and significance of the poly(T) motif in our\nstudy.\n10 30 90\nComment 2: To properly interpret the data presented in Fig. 1a–g, the efficiencies of HSV-1 HF, HSV-1 F, and HSV-1 KOS\ninfection in BMDMs need to be shown. The authors should analyze the expression levels of representative immediate-early,\nearly, and late HSV-1 proteins in these infected cells at various times after infection. Moreover, the authors should examine\nthe expression levels of VP22, which has been reported to inhibit AIM2 inflammasome activation, in infected cells at various\ntimes after infection.\nThank you for this important suggestion. To evaluate and compare the infection\nefficiencies of HSV-1 HF, F, and KOS strains, we performed time-course infection\nexperiments in HaCaT cells, a human keratinocyte line relevant to HSV-1 pathogenesis.\nCells were infected with each strain at the same multiplicity of infection (MOI = 3), and\nsamples were collected at multiple time points (3, 6, 9, and 12 hours post-infection). We\nthen assessed the expression of representative HSV-1 viral proteins across different\nphases of the viral life cycle using immunoblotting: ICP0, ICP8, gD, gC, and VP22.\nOur results showed that all three viral strains (HF, F, and KOS) successfully infected both\nHaCaT cells, with comparable expression levels of ICP0, ICP8, gD, gC, and VP22 over time.\nThe kinetics and intensity of viral protein expression were similar across strains,\nsuggesting that differences in AIM2 activation observed in our study were not due to\nvariations in viral entry, replication efficiency, or protein expression levels.\nIn addition, to directly address your concern in a macrophage system relevant to\ninflammasome activation, we performed parallel infection experiments using primary wild-\ntype (WT) and Aim2−/− bone marrow–derived macrophages (BMDMs). Primary WT and\nAim2−/− BMDMs were infected with HSV-1 HF, F, or KOS strains at a high multiplicity of\ninfection (MOI = 10), and viral protein expression was analyzed at 12 hours post-infection.\n12\n1PSAC\nWT Aim2–/–\n****\nC 10 30 90 C mer 400 WT\nP45 300 Aim2–/–\n200\n100 ns ns ns\nP20\nActin 0\n)lm/gp(β1-LI\nControl 10 mer 30 mer 90 mer\nImmunoblot analysis demonstrated that representative immediate-early (ICP0), early\n(ICP8), and late (gD and VP22) viral proteins were expressed at comparable levels across\nall three HSV-1 strains in both WT and Aim2−/− BMDMs. These results indicate that, even\nunder conditions optimized for inflammasome activation in primary macrophages, HSV-1\nHF, F, and KOS establish infection with similar efficiency and comparable viral protein\nexpression profiles. Therefore, the strain-specific differences in AIM2 inflammasome\nactivation observed in our study cannot be explained by differential viral infection\nefficiency or viral protein expression in BMDMs.\nImportantly, VP22 was robustly expressed in all three strains and at all examined time\npoints, indicating that the differential inflammasome activation cannot be attributed to the\nabsence or delay in VP22 expression in the F or KOS strains.\nThese new data have been included in the revised manuscript (New Data, Extended Data\nFigure 1a, 1b [shown below]) and described in the Results section. We believe this analysis\naddresses your concern and reinforces the conclusion that the enhanced AIM2 activation\nby the HF strain is due to its unique poly(T) DNA content rather than differences in infection\nefficiency.\nHSV-1(HaCaT)\na b\nKOS F HF\nM 3 6 9 12 3 6 9 12 3 6 9 12 :hpi\nICP0\nICP8\ngD\nVP22 VP22\ngC\nGAPDH\nComment 3: Fig. 2 a–g. The effects of AIM2 knock-out on the expression of VP22 in BMDMs\ninfected with each of the HSV-1 strains should be analyzed.\nThank you for raising this important point. To address your concern regarding the effect\nof AIM2 deficiency on VP22 expression, we performed parallel infection experiments using\nprimary wild-type (WT) and Aim2−/− bone marrow–derived macrophages (BMDMs). Primary\nWT and Aim2−/− BMDMs were infected with HSV-1 HF, F, or KOS strains at a high multiplicity\nof infection (MOI = 10), and viral protein expression was analyzed at 12 hours post-\ninfection. Immunoblot analysis revealed that the expression levels of VP22 were\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nHSV-1(pBMDMs)\nMock HF F KOS\nICP0\nICP8\ngD\nActin\ncomparable between WT and Aim2−/− BMDMs for all three HSV-1 strains. Consistently,\nother representative viral proteins, including the immediate-early protein ICP0, the early\nprotein ICP8, and the late protein gD, also showed similar expression patterns irrespective\nof AIM2 status. These results indicate that AIM2 deficiency does not affect VP22\nexpression during HSV-1 infection in BMDMs. Therefore, the differences in AIM2\ninflammasome activation observed in our study cannot be attributed to altered VP22\nexpression resulting from AIM2 knock-out, but rather reflect downstream sensing of viral\nligands.\nThese new data have been included in the revised manuscript (New Data, Extended Data\nFigure 1b [shown below]) and described in the Results section.\nb\nVP22\nComment 4: Fig. 3 c–e. Based on these results, the authors concluded that VP22 did not inhibit\nHSV-1-HF-induced AIM2 inflammasome activation. However, the data do not include the\nexpression levels of VP22 in transfected BMDMs, and therefore, it is uncertain whether sufficient\nVP22 was expressed in these cells. The authors should analyze whether transfection with the\nVP22 expression vector increased the levels of VP22 in the cells. Moreover, the authors should\nexamine the effects of the VP22-null mutation on AIM2 inflammasome activation (caspase-1\ncleavage, IL-18, and IL-1β secretion, and cell death) in BMDMs using VP22-null mutant HSV-1\nHF together with VP22-null mutant HSV-1 F or KOS as a control.\nWe appreciate this thoughtful and constructive comment. To address the first point, we\nperformed immunoblotting to confirm VP22 expression following transfection of the VP22\nexpression vector. In mock-infected BMDMs, VP22 was expressed at detectable levels\nupon transfection, confirming successful overexpression. Moreover, subsequent infection\nwith HSV-1 HF did not further enhance VP22 expression. Importantly, VP22 expression\nlevels were comparable between WT and Aim2−/− BMDMs under all conditions tested (New\nData, Figure 3b [shown below]). These results indicate that VP22 expression in primary\nmacrophages is not readily augmented by exogenous overexpression or viral infection\nand is not influenced by AIM2 deficiency. Importantly, even under these physiologically\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nHSV-1(pBMDMs)\nMock HF F KOS\nICP0\nICP8\ngD\nActin\nrelevant conditions, HF infection robustly activated the AIM2 inflammasome, supporting\nour conclusion that VP22 does not inhibit HF-induced AIM2 inflammasome activation.\nb\nVP22\nActin\nTo further investigate the role of VP22 in AIM2 inflammasome regulation, we generated\nVP22-null mutant viruses in both the HF and F strain backgrounds using CRISPR/Cas9-\nmediated genome editing (New Data, Extended Data Figure 5a, 5b [shown below]).\nMedia\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nControl\n–/–2miA\nVP22\nHF Media HF\na HSV-1 genome\nU U\nL S\nTRl VP22 IRl IRs TRs\ngRNA\nViral DNA VP22\neGFPdonor\nHAS2 eGFP CMVp HAS1\ntemplate\nPoly A\nb\nF1 F2\nWT VP22\nR1 R2\nF1 F2\nΔVP22 eGFP CMVp\nPoly A R1 R2\nHF F\n2 2 2 2\n2 2 2 2\nP P P P\nTV TV TV TV\nWΔ WΔ WΔ WΔ\n2 kb - 2 kb -\n1 kb -\n1 kb -\nF1+R1 F2+R2 F1+R1 F2+R2\nSuccessful deletion of VP22 was confirmed by immunoblot analysis, which showed\ncomplete loss of VP22 protein expression in the mutant viruses. Comparison of viral\nprotein expression between WT and VP22-null viruses revealed that overall viral protein\nlevels were slightly reduced in the VP22-null mutants compared with WT viruses,\nconsistent with the known role of VP22 in optimal viral fitness (New Data, Extended Data\nFigure 5c, 5d [shown below]).\nHSV-1 HF HSV-1 F\nc d\nWT ∆VP22 WT ΔVP22\nM 3 6 9 3 6 9 (hpi) M 3 6 9 3 6 9 (hpi)\nICP0 ICP0\nICP8 ICP8\nVP22 VP22\ngC\ngC\nGAPDH\nGAPDH\nIn the HF background, BMDMs infected with WT HF or VP22-null HF showed comparable\nlevels of caspase-1 cleavage and IL-1β and IL-18 secretion (New Data, Figure 3h, 3i, 3j\n[shown below]). These results indicate that deletion of VP22 does not alter AIM2\ninflammasome activation in the HF strain, supporting our conclusion that VP22 is not a\ncritical regulator of inflammasome activation in this context. Accordingly, the enhanced\nAIM2 activation observed during HF infection is most parsimoniously explained by HF-\nspecific genomic features, such as the long poly(T) DNA sequence.\n16\n)lm/gp(\nβ1-LI\n****\n120\n90\n60\n30\nWT\nTW\n–/–2miA\nns\n150\n****\nTW\n–/–2miA\nHF\nDVP22\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\nHF HF\nWT DVP22\nActin\n–/–2miA\n)lm/gn(\n81-LI\n2.0 ****\n1.5\n1.0\n0.5\nWT\nTW\n–/–2miA\nns\n****\nTW\n–/–2miA\nh i j\nHF\nDVP22\nIn contrast, a distinct outcome was observed in the F strain background. While WT F virus\nfailed to induce detectable caspase-1 activation or IL-1β and IL-18 secretion, infection with\nthe VP22-null F mutant robustly induced these inflammasome-associated readouts (New\nData, Extended Data Figure 5e. 5f. 5g [shown below]). These findings demonstrate that,\nunlike HF, AIM2 inflammasome activation in the F strain is VP22-dependent. This result is\nconsistent with previous reports showing that VP22 suppresses inflammasome activation\nin HSV-1 F and related strains (PMID: 29447697).\nTaken together, our data reveal a strain-dependent role of VP22 in regulating AIM2\ninflammasome activation. While VP22 functions as an inhibitory factor in F strain infection,\nit is dispensable for inflammasome regulation in the HF strain, where viral DNA features\nsuch as poly(T) DNA play a dominant role. These new data have been included in the\nrevised manuscript in the Results section.\nComment 5: The authors should show whether the 23-mer poly(T) sequence is conserved in other\nlaboratory strains and clinical isolates of HSV-1. Genetic modification (i.e., mutations associated\nwith syncytial phenotype) sometimes artificially occurs during serial passages in cell cultures.\nHow did the authors eliminate this possibility for the 23-mer poly(T) sequence in the HF strain?\nThank you for this insightful comment. To determine whether the 23-mer poly(T) sequence\nidentified in the HF strain is the result of artificial mutation during laboratory passage or\nrepresents a naturally occurring variant, we conducted a comprehensive analysis of\npublicly available HSV-1 and viral genome sequences.\nFirst, we examined 492 complete HSV-1 genome datasets from the NCBI database. Among\nthese, 18 sequences contained a 23-mer poly(T) sequence, including several well-\ncharacterized laboratory strains (e.g., H166, KOS63, ZW63) and multiple clinical isolates\n(e.g., from Greninger, A.L.; Rathbun, M.; Furlong, J.; Johnston, C.). These data indicate\nthat the 23-mer poly(T) sequence is not unique to HF or lab-adapted strains but is also\npresent in naturally circulating viral populations (New Data, Extended Data Figure 10a\n[shown below]).\n17\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\ne F F\nWT DVP22\nActin\n)lm/gp(\nβ1-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n200 ****\nTW\n–/–2miA\nns\nF\nDVP22\n)lm/gp(\n81-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n****\n–/–2miA\nf g\n250\nns\nF\nDVP22\n80\n75.0%\n70\n60 61.1% Clinical sample\nKOS63\n30 27.7% Others\n20\n13.1%\n10 8.3%\n5.5% 5.5%\n2.3% 1.1%\n23 mer 14 mer\nAdditionally, we found that a 14-mer poly(T) sequence, which we used as a reference for\nthe ΔT mutant, was present in approximately 15% (63/426) of clinical HSV-1 samples,\n14\nwhile the 23-mer poly(T) sequence appeared in 3% (11/426) of those clinical datasets (New\nData, Extended Data Figure 10b [shown below]). This pattern of distribution supports the\ninterpretation that poly(T) sequence length variability is a naturally occurring\npolymorphism among HSV-1 strains, rather than a cell culture–induced artifact such as\nthose seen in syncytial mutations.\n18\negatnecreP\n%\na\nb\n100\n80\n60\n40\n20\n0\not\nal\nS a m\npl e\nH 1\n6 6\nK O S\n6 3\nR\nH 2\nni c\nal\ns a m\npl e\nOt h\ner s\nT Cli\nnoitroporP\n%\n100% 100%100%\n78%\nPoly(T) 14 mer\nPoly(T) 23 mer\n56%\n50%\n20%\n17% 15%\n4% 3% 0%\nTo further explore the biological relevance of these poly(T) tracts, we conducted a broader\ncross-species sequence analysis using the NCBI viral genome repository. Among the DNA\nviral genome datasets containing a 23-mer poly(T) sequence, we found that about 90% are\nfrom Monkeypox virus, Human herpesvirus and adenovirus. Among the RNA viral genome\ndatasets containing a 23-mer poly(T) sequence, we found that about 90% are from\nHepacivirus and SARS-Coronavirus (New Data, Supplementary Table 1).\nFor the 14-mer poly(T) motif, in addition to Monkeypox virus, we detected matches in\nHuman herpesvirus 4 (EBV), Human papillomavirus 16 (HPV16), and Human\nmastadenovirus D (HAdV-D) among DNA viruses. Among RNA viruses, we identified\npoly(T)-containing sequences in Hepacivirus, SARS-CoV-2, and Classical Swine Fever\nVirus (CSFV).\nThese findings indicate that long poly(T) tracts are not exclusive to HSV-1, but rather are\nconserved across a broad spectrum of clinically important DNA and RNA viruses. This\nwidespread occurrence suggests that poly(T) sequences may play a previously\nunderappreciated role in innate immune sensing, and further supports the relevance of\nour findings in a broader virological and immunological context.\nIn conclusion, both intra-species HSV-1 analysis and cross-species comparisons strongly\nrefute the idea that the 23-mer poly(T) sequence is a culture-derived artifact. Instead, the\ndata support the view that this sequence represents a naturally occurring, functionally\nrelevant genetic motif that may shape strain-specific host responses via AIM2\ninflammasome activation.\nComment 6: Characterization of HFdelata14 is required. The authors should examine the effects\nof deltaT14 on viral replication, infection efficiency, the expression levels of neighboring genes\n(UL20 and UL21), and VP22 expression levels in BMDMs. To eliminate the off-target effect(s) of\nCRISPR/Cas9-mediated genome editing, the authors should construct a recombinant virus in\nwhich the mutation in HFdelta14 is repaired and test it together with wild-type and HFdelta14\nviruses in the experiments shown in Fig. 6 and Extended Data Fig. 4.\nThank you for this critical and constructive comment. In response, we performed a series\nof additional experiments to comprehensively characterize the HFΔT mutant and to\n14\nassess whether the observed phenotypes were due to the targeted deletion of the 14-bp\npoly(T) motif or potential off-target effects.\nFirst, to evaluate infection efficiency and viral replication, we infected WT and Aim2−/−\nBMDMs with WT HF, HFΔT , and the revertant virus (HFΔT -Rev), in which the deleted 14-\n14 14\nbp poly(T) motif was reintroduced via homologous recombination (New Data, Extended\nData Figure 7a, 7b [shown below]).\n19\na HSV-1 genome\nU U\nL S\nTRl UL25 UL26 IRl IRs TRs\nSelected plaques\ngRNA#1\nUL25 TTT············TT UL26 Bright field eGFP\neGFPdonor Step1\ngRNA#2\ntemplate\nHAS1 CMVp eGFP IRES HAS2\nHF ΔT eGFP-IRES-UL26\n27\n(intermediate)\nPoly(T) donor Step2\ntemplate\nHAS1 TTT··TT HAS2\nb Sequencing HF ΔT 14\nHF ΔT HF ΔT R F T F T R\n14 14 27 27\nforward strand reverse strand reverse strand forward strand\nWe then examined the expression of viral proteins ICP0 (immediate-early), ICP8 (early), gD,\nand VP22 (tegument) at 12 hours post-infection. All three viruses showed comparable\nprotein expression levels regardless of host genotype (WT vs. Aim2−/−) (New Data,\nExtended Data Figure 1c [shown below]), indicating that the poly(T) deletion does not\nimpair viral gene expression or infectivity.\nc\nICP0\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nHF\nHF HF DT\nVP22\ngD\nActin\nMoreover, we assessed the expression levels of the neighboring genes, UL25 and UL26,\nusing qPCR, and found no significant differences among the three virus groups (New Data,\nExtended Data Figure 7e [shown below]), confirming that the deletion does not disrupt\nadjacent loci.\ne\n2.0\n1.5\n1.0\n0.5\n0.0\nW T D T14 DT1 R 4\nTo directly address the concern regarding off-target effects of CRISPR/Cas9 editing, we\ngenerated the HF revertant virus by precisely restoring the deleted poly(T) sequence in the\nHFΔT genome. We then tested all three viruses, WT HF, HFΔT , and HFΔT -Rev, in the\n14 14 14\nsame experimental settings presented in Figure 6 and Figure 7.\nIn in vitro experiments, the results were obtained using primary BMDMs. HFΔT -Rev\n14\ninfection led to robust caspase-1 cleavage, IL-1β and IL-18 secretion, and pyroptotic cell\ndeath, restoring the inflammatory responses to levels comparable with those induced by\nWT HF (New Data, Figure 6a–e). These responses were again significantly attenuated in\nHFΔT -infected cells. Importantly, in Aim2−/− BMDMs, none of these inflammasome\n14\nresponses were detected, clearly demonstrating that inflammasome activation by the\npoly(T)-containing HF strain is entirely dependent on AIM2 in cellular level.\n21\n)dlof(\nANRm52LU\n2.0\n✱✱\nns 1.5\n1.0\n0.5\n0.0\nW T D T14 T1 R 4 D\n)dlof(\nANRm62LU\nHSV-1 HF\nns\nns\nIn in vivo experiments, the HFΔT -Rev strain fully rescued the survival phenotype,\n14\nrestoring host protection to levels comparable to those observed in WT HF–infected mice\n(New Data, Figure 7a, 7b [shown below]). Furthermore, lung viral titers, caspase-1\nactivation, and proinflammatory cytokine production (IL-1β and IL-18) were significantly\nrestored in mice infected with HFΔT -Rev, closely resembling the responses observed in\nwere markedly reduced in mice infected with the HFΔT mutant (New Data, Figure 7a–7g\n14\n[shown below]), indicating a loss of inflammasome activation and antiviral defense.\nNotably, in Aim2−/− mice, all inflammasome-associated responses, including caspase-1\nactivation and cytokine production, were completely abolished regardless of virus strain\n(New Data, Figure 7a–7g [shown below]), confirming that the observed immune\nphenotypes are strictly AIM2-dependent in vivo.\nMedia\n–/–2miA\nTW\n–/–2miA\nHF\nWT\n1PSAC\nTW\n–/–2miA\nTW\n–/–2miA\nHF\nHF DT\n120\n90\nActin\nHF\nWT\nTW\n–/–2miA\nTW\n–/–2miA\nDT\n14\n)lm/gp(β1-LI\n****\nns\nTW\n–/–2miA\n****\n****\nHF\nDT\n)lm/gn(\n81-LI\n2.0\n1.5\n1.0\n0.5\nWT\nTW\n–/–2miA\nns\n****\nTW\n–/–2miA\nh i j\nHF\nDT\n–/–2miA\n****\n****\nHF\nDT\n100\n75\n50\n25\n0\nhtaed\nllec\n%\n)sllec+xotyS(\nHF\nWT\nTW\n–/–2miA\nTW\n–/–2miA\nHF\nDT\n–/–2miA\na b c\nd e\n****\nHF WT HF DT HFDT Rev **** ****\n14 14\nWT Aim2–/– WT Aim2–/– WT Aim2–/–\nns\nHF\nDT\n100\n80\n60\n40\n20\n14\nspecifically attributable to the loss of the 14-mer poly(T) sequence, rather than off-target\neffects. The successful rescue of inflammasome activation and protective immunity by the\nrevertant virus provides strong genetic evidence supporting the functional importance of\nthe poly(T) motif in driving AIM2 inflammasome activation.\nWe have incorporated these new results into the revised manuscript and believe they\ndirectly and fully address your concern regarding specificity and genome integrity\nfollowing genome editing.\nComment 7: To further verify the authors’ conclusion that the length of the poly(T) sequence in\nthe intergenic region of HSV-1 genome is critical for AIM2 inflammasome activation during HSV-\n1 infection, the authors should construct a recombinant virus derived from HSV-1 F or a KOS strain harboring the 27-mer poly(T) sequence in the intergenic region, as well as its repaired virus,\n23\n)%(\nlavivruS\nWT mice\nns\nHF WT(n = 18)\nHF DT (n = 20) 14\n**** HFDT 14 Rev(n = 20)\n0 2 4 6 8 101214\nDays post-infection\n100\n80\n60\n40\n20\n0\n)%(\nlavivruS\nAim2–/– mice\nHF WT(n = 21)\nHF DT (n = 24) 14\nns HFDT Rev(n = 24)\n14\n0 2 4 6 8 101214\nDays post-infection\nTW\n–/–2miA\nTW\n–/–2miA\nW T T14\nH F H F\nD\nTW\n–/–2miA\n300\n200\n100\n0\nv e R\nT14 D\nF\nH\n)lm/gp(β1-LI\n****\n**** ****\nns\nTW\n–/–2miA\nTW\n–/–2miA\nW T T14\nH F H F\nD\nTW\n–/–2miA\n****\n1500 **** ****\n1000\n500 ns\n0\nv e R\nT14 D\nF\nH\nTW\n–/–2miA\nTW\n–/–2miA\n)lm/gp(\n81-LI\nW T T14\nH F H F\nD\nTW\n–/–2miA\n****\n**** ****\nns\nv e R\nT14 D\nF\nH\n)eussit\ng/UFP(1VSH\nHF WT\n5 x 107\n4 x 107\n3 x 107\n2 x 107\n1 x 107\n0\n1PSAC\nHF DT 14\nWT Aim2–/–WT Aim2–/–\nP45\nP20\nActin\nHF WT HFDT Rev\n1PSAC\nc\na\nb\nd\nP45\nP20\nActin\ne f g\nand test them together with wild-type HSV-1 F or KOS in the experiments in Fig. 6 and Extended\nData Fig. 4, after characterization of the recombinant viruses, as described in Comment 6.\nThank you for this excellent suggestion, which has helped us further validate the critical\nrole of the poly(T) sequence in AIM2 inflammasome activation. In response, we generated\na recombinant HSV-1 F strain harboring a 27-mer poly(T) sequence inserted into the UL25–\nUL26 intergenic region (F T ), as well as a corresponding revertant virus (F T -Rev) in\n27 27\nwhich the poly(T) sequence was precisely removed via homologous recombination.\nTo assess the functional consequences of the poly(T) insertion, we performed in vivo\ninfection experiments in both WT and Aim2−/− mice. We found that in WT mice, infection\nwith the F T virus significantly improved survival compared to the parental F or F T -Rev\n27 27\nstrains, which induced rapid and severe mortality (New Data, Extended Data Fig. 8b, 8c).\nIn contrast, in Aim2−/− mice, all three viral strains caused uniformly lethal infections,\nregardless of the presence or absence of the poly(T) sequence. These findings clearly\ndemonstrate that the poly(T)-mediated protection is AIM2-dependent.\n100\n80\n60\n40\n20\n27\nbut not in those infected with WT F or F T -Rev strains. Again, these responses were\n27\ncompletely abolished in Aim2−/− mice (New Data, Extended Data Fig. 8d, 8e), further\nconfirming that the inserted 27-mer poly(T) sequence was sufficient to activate the AIM2\ninflammasome in vivo.\n24\n)%(\nlavivruS\nWT mice\n100\n80\n**** F WT(n = 12)\nns F T Rev(n = 14) 20\n27\n0\n0 2 4 6 8 10 1214\nDays post-infection\n)%(\nlavivruS\nb c\nAim2–/– mice\nF WT(n = 8)\nF T (n = 11) 27\nns F T Rev(n = 11)\n27\n0 2 4 6 8 10 1214\nDays post-infection\nWT Aim2–/–WT Aim2–/–\n1PSAC\nF T F T Rev\n27 27\nWT Aim2–/–WT Aim2–/–\nP45\nP20\nActin\n1PSAC\nd e\nWT F F T\nP20\nActin\nIn in vitro studies using primary BMDMs, infection with the F T virus led to strong\n27\ncaspase-1 cleavage, whereas WT F and F T -Rev strains failed to activate these pathways\n27\n(New Data, Extended Data Fig. 8a). Importantly, inflammasome activation was completely\nabsent in Aim2−/− BMDMs, once again confirming the strict dependence on AIM2 sensing.\nTo ensure that these phenotypic differences were not due to variations in viral replication\nor fitness, we examined the expression levels of representative viral proteins including\nICP0 (immediate-early), ICP8 (early), gD and gC (late glycoproteins), and VP22 (tegument).\nImmunoblot analysis revealed comparable levels of viral protein expression across WT F,\nF T , and F T -Rev strains, indicating that the observed immune phenotypes were not\n27 27\nattributable to differences in viral infectivity or gene expression (New Data, Extended Data\nFig. 1d).\nMoreover, we assessed the expression levels of the neighboring genes, UL25 and UL26,\nusing qPCR, and found no significant differences among the three virus groups (New Data,\nExtended Data Figure 7f [shown below]), confirming that the deletion does not disrupt\nadjacent loci.\nMedia\n–/–2miA\nTW\n–/–2miA\nF\nWT\n1PSAC\nTW\n–/–2miA\nTW\n–/–2miA\na F\nF T\nP20\nActin\nd\nICP0\nICP8\nVP22\ngD\nActin\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nHSV-1 F\nMock WT T T Rev\n27 27\n2.0\n1.5\n1.0\n0.5\n0.0\nW T T27\nT2\nR\nmer poly(T) sequence is sufficient to confer AIM2 inflammasome activation, and that this\nresponse translates into enhanced antiviral defense and survival in vivo. These findings\nfurther reinforce the conclusion that the poly(T) sequence length in the HSV-1 genome is\na key determinant of strain-specific AIM2 activation.\nComment 8: All analyses were conducted using mouse-derived cells. Since HSV-1 is a human\npathogen, the authors should perform representative experiments shown in Figs. 2–5 and\nExtended Data Fig. 4 using human-derived cells.\nThank you for this important suggestion. Recognizing that HSV-1 is a human-specific\npathogen, we extended our analysis to a human cellular system by performing\nrepresentative validation experiments in THP-1–derived human macrophages, which are\nwidely used for studying inflammasome biology.\nTo specifically assess the role of AIM2, we performed siRNA-mediated knockdown of AIM2\nin PMA-differentiated THP-1 macrophages (New Data, Extended Data Fig. 9). We first\ncompared WT HF and the HF VP22-null mutant in both WT and siAIM2 THP-1 cells. In WT\ncells, both viruses induced comparable levels of cell death, suggesting that VP22 does not\nsignificantly suppress AIM2 activation in the HF background, consistent with our findings\nin murine BMDMs. Importantly, this HF-induced cell death was abolished in siAIM2-treated\ncells, confirming the AIM2-dependence of the response in human macrophages (New Data,\nExtended Data Fig. 9a, 9b).\nWe next compared WT HF, HF ΔT , and the HFΔT -Rev viruses. In WT THP-1 macrophages,\n14 14\nHFΔT -Rev restored the cell death response to levels similar to WT HF, while the ΔT\n14 14\nmutant failed to induce significant cell death. This pattern was again reversed by siAIM2\nknockdown, indicating that the poly(T)-dependent activation of AIM2 is conserved in\nhuman cells (New Data, Extended Data Fig. 9c, 9d).\n26\n)dlof(\nANRm52LU\n2.0\nns\n✱ 1.5\n1.0\n0.5\n0.0\nW T T27\nT2\nR\n7\n)dlof(\nANRm62LU\nf\nHSV-1 F\nns\nns\nFinally, we tested WT F, F T , and the F T -Rev viruses. As expected, the F-T virus\n27 27 27\ninduced strong AIM2-dependent cell death in WT THP-1 cells, whereas both WT F and F-\nT -Rev failed to do so. In AIM2-silenced cells, all strains failed to trigger cell death,\n27\nconfirming that the poly(T)-mediated activation of inflammasome signaling is specifically\ndependent on AIM2 (New Data, Extended Data Fig. 9e, 9f).\nAcross all tested viral conditions, including VP22 deletion, poly(T) insertion/deletion, and\nstrain background, we observed that the inflammasome activation phenotypes observed\nin murine BMDMs were faithfully recapitulated in human macrophages, and that AIM2 was\nessential for these responses in both systems.\nThese findings provide strong evidence that our conclusions regarding poly(T)-mediated\nAIM2 activation and the non-essential role of VP22 in the HF background are applicable to\nhuman innate immune contexts. We have incorporated these new data into the revised\nmanuscript (New Data, Extended Data Fig. 9) and extended the Discussion to emphasize\nthe translational relevance of our findings in the human setting.\nWT siAIM2 WT siAIM2 WT siAIM2\nHF WT HF DT HF DT Rev\n14 14\nWT siAIM2 WT siAIM2 WT siAIM2\nF WT F T F T Rev\n27 27\nWT siAIM2 WT siAIM2 WT siAIM2\n****\nHF\nWT\nMinor Comments\nComment 1: The ASC-citrine specks in Fig. 1a and Fig. 2a are unclear. Enlarged or higher-\nresolution images should be provided to clearly delineate the ASC-citrine specks.\nThank you for pointing out the need for higher-resolution imaging to better visualize ASC-\ncitrine specks. The original images presented in Figures 1a and 4a were acquired using\nthe IncuCyte live-cell imaging system, which is optimized for real-time monitoring of speck\n2MIAis\nns\n****\nTW\n2MIAis\n80\n60\n40\n20\nDVP22\nhtaed\nllec\n%\n)sllec+xotyS(\n100\n75\n50\n25\n0\nhtaed\nllec\n%\n)sllec\n+xotyS(\nHF\nWT\nTW\n2MIAis\nTW\n2MIAis\nHF\nDT\n2MIAis\n**** 100\n**** **** 75\n50\nns 25\nDT\nhtaed\nllec\n%\n)sllec\n+xotyS(\nF\nWT\nTW\n2MIAis\nTW\n2MIAis\nF\nT\n2MIAis\na\nc\ne\nb d f\nns\n****\n****\nns ns\nF\nT\nkinetics but offers limited spatial resolution for fine structural features. As a result, we\nagree that the visualization of ASC specks in these images was suboptimal.\nIn response to this concern, we performed new imaging using high-resolution confocal\nmicroscopy (Zeiss LSM 900) to provide clearer and more detailed visualization of ASC\nspeck formation. These confocal images were acquired using z-stack projection and high-\nmagnification settings, allowing us to resolve individual ASC specks with much greater\nclarity and precision.\nAccordingly, we have removed the original IncuCyte-derived images from Figures 1a and\n4a and replaced them entirely with confocal images. We believe this revision addresses\nyour concern and significantly improves the quality and interpretability of the imaging data\nin our manuscript.\nHF F KOS\nComment 2: The authors should disclose detailed information about the VP22 expression plasmid,\nthe gRNA sequences, and the names of the gRNA expression plasmids for the knockout iBMDMs,\nas well as sequence information for each gene after genome editing.\nThank you for this important and practical suggestion. In response, we have now provided\ncomprehensive information regarding all constructs and reagents used in this study,\nincluding:\n1) VP22 expression plasmid: Detailed information on the VP22 expression construct\nhas been added to the revised Methods section. Specifically, the F strain VP22\nexpression vector (pLV[Exp]-EGFP:T2A:Puro-EF1A>VP22) was purchased from\nVectorBuilder (VB220920-1383rfw). The construct encodes VP22 under the control\nof the EF1A promoter and includes an EGFP:T2A:Puro cassette for expression\ntracking and selection. BMDMs were transfected with the VP22 expression plasmid\nusing DOTAP (Roche, 11202375001) 12 hours prior to HSV-1 infection, as\nTW\nMock\nHF DNA KOS DNA\nMDMB\nTW\nMock\ndescribed in the Methods. These details clarify the source, design, and\nexperimental use of the VP22 expression plasmid in this study.\n2) gRNA sequences and plasmid names: For each gene knockout generated in\nimmortalized BMDMs (iBMDMs), we have now provided the complete gRNA\nsequence information together with the names and catalog numbers of the\ncorresponding gRNA expression plasmids used for CRISPR/Cas9-mediated\ngenome editing. As described in the revised Methods section, Sting−/−, cGas−/−,\nMyd88−/−, and Trif−/− iBMDMs were newly generated in this study by electroporation\nof WT iBMDMs with a Cas9-encoding plasmid (Addgene #43945) and individual\ngRNA expression plasmids (Addgene #104174) using the Amaxa 4D-Nucleofector\nsystem. All gRNA sequences and plasmid details are now explicitly listed in Source\ndata 1 [shown below] and are referenced in the “Generation of knockout clonal\ncells” subsection of the Methods, thereby ensuring transparency and\nreproducibility of the genome-editing procedures.\nCasp1-/-\nGAGGAAGCAATTTATCAACTCAGTGAGTATAGGGACAATAAATGGATTGTTGGATGAACTTTTAGAGAAGAGAGTGCTGAATCAGGAA Reference\nGAGGAAGCAATTT-------------------------------------------------TAGAGAAGAGAGTGCTGAATCAGGAA -49 bp\nCasp3-/-\nGTTGACTAGCTTCTTCAGAGGCGACTACTGCCGGAGTCTGACTGGAAAGCCGAAACTCTTCATCATTCAGGTAGGGCATTGCCCAGCA Reference\nGTTGA-----------------------------------------------------------------------------CCAGCA -77 bp\nCasp6-/-\nTGCGAGTCAGGTTGTCTCTGTCGCGTTGGTGCCCCGCCTCTCTGGGAGGGTCAGGTGCCAAAAGAACCTCTCGTGATTGAAGATGAGG Reference\nTGCGAGTCAGGTTGTCTCTGTCGCGTTGGTGCCCCGC----CTGGGAGGGTCAGGTGCCAAAAGAACCTCTCGTGATTGAAGATGAGG -4 bp\nCasp7-/-\nCGGTACAGATAAGTGGGCACTCGGTCCCGGCCGGTCCTGACGGGGCCCGCAGAGGCATTTCTCTTCTTCTTCCTTGGGTTACAGAAAG Reference\nCGGTACAGATAAGTGGGCACTCGGTCCCGGCCGGTCCT-ACGGGGCCCGCAGAGGCATTTCTCTTCTTCTTCCTTGGGTTACAGAAAG -1 bp\nCasp9-/-\nCACATGAGGACTCACCATATCTGCATGTCCCCTGATCTTCCCTGGAACACCTGAGAAAACAGGAGAAAGAAGAAATGTGGGTGTTGAG Reference\nCACATGAGGACTCACCATATCTGCATGTCCCCTGATCT-CCCTGGAACACCTGAGAAAACAGGAGAAAGAAGAAATGTGGGTGTTGAG -1 bp\nGsdme-/-\nAAGTGTGAGAACCATAAGAGCGGGGCTATTGGGACAGTC-GTGGGGAAGGTCAAGCTGAACGTTGGTGGCAAAGGCGTGGTGGAGAGT Reference\nAAGTGTGAGAACCATAAGAGCGGGGCTATTGGGAC------------AGGTCAAGCTGAACGTTGGTGGCAAAGGCGTGGTGGAGAGT -11 bp\nAAGTGTGAGAACCATAAGAGCGGGGCTATTGGGACAGTCCGTGGGGAAGGTCAAGCTGAACGTTGGTGGCAAAGGCGTGGTGGAGAGT +1 bp\nTmlkl-/-\nTCTGCTGGTTAGCCTCCTTCAGGACTTCATCAAAACGGCCCAGGGCAGCAGTAATGTCATCGGGCAGGTTCTTCTTTCCTTGGGCCTG Reference\nTCTATCTGCTGGTTAGCCTCCT--------------------------------------TCATCGGGCAGGTTCTTCTTTCCTTGGG -38 bp\nTCTGCTGGTTAGCCTCCTTCAGGACTTCATCAAAACGG-CCAGGGCAGCAGTAATGTCATCGGGCAGGTTCTTCTTTCCTTGGGCCTG -1 bp\nRipk3-/-\nGAAGAACCTTTCTCAGGCCTACTGGTGCGTCAGCGGTTCCTCTGGTGAGCCGTGAAGAACTGAAGAAGCTGGAGTTTGTGGGTAAAGG Reference\nGAAGAACCTTTCTCAGGCCTACTGGTGCGTCAGCGGTT-CTCTGGTGAGCCGTGAAGAACTGAAGAAGCTGGAGTTTGTGGGTAAAGG -1 bp\nRipk3-/-Caps8-/-\nRIPK3\nGAGCAAGAGAGAATGAAGAATGGGTACCTGTCATTGGATTCGGTGGGTCCAGGGATACCAAGGAGTGCCGTGTCTTCCATCTCCCTGC Reference\nGAGCAAGAGAGAATGAAGAATGGG------------------------------ATACCAAGGAGTGCCGTGTCTTCCATCTCCCTGC -30 bp\nCasp8\nCGGAATATAGTTCTTGTGAGATGATGAATCCACTTCTAAAGTGTGGTTCTGTTGCTCGAAGCCTGCCTCATCAGGCACTCCTTTCTGG Reference\nCGGAATATAGTTCTTGTGAGATGATGAATCCACTTCTAA-GTGTGGTTCTGTTGCTCGAAGCCTGCCTCATCAGGCACTCCTTTCTGG -1 bp\nCGGAATATAGTTCTTGTGAGATGATGAATCCACTT-----GTGTGGTTCTGTTGCTCGAAGCCTGCCTCATCAGGCACTCCTTTCTGG -5 bp\nAIM2-/-\nCTTTTCAGGCTGATCCTGGGACTGTGAGATGGAGAGTGAGTACCGGGAAATGCTGTTGTTGACCGGCCTGGACCACATCACGGAGGAAGA Reference\nCTTTTCAGGCTGATCCTGGGACTGTGAGATGGAGAGTG----CCGGGAAATGCTGTTGTTGACCGGCCTGGACCACATCACGGAGGAAGA -4 bp\nCTTTTCAGGCTGATCCTGGGACTGTGAGATGGAGAGTGAG-ACCGGGAAATGCTGTTGTTGACCGGCCTGGACCACATCACGGAGGAAGA -1 bp\ncGAS-/-\nGCCATCTTTCACATGTGGACCCAGGACCCGCAGGACAGTCAGTGGGACCCCAGGAACCTCAGCTCCTGCTTCGATAAGTTGTTAGCATTC Reference\nGCCATCTTTCACATGTGGACCCAGGACCCGCAGGACAA--AGTGGGACCCCAGGAACCTCAGCTCCTGCTTCGATAAGTTGTTAGCATTC -2bp\nGCCATCTTTCACATGTGGACCCAGGACCCGCAGGACAG--AGTGGGACCCCAGGAACCTCAGCTCCTGCTTCGATAAGTTGTTAGCATTC -2bp\nIRF1-/-\nTAACACTCACTTTATTGATCCAGATCAGCCCTGGGATTTGGTTGGAATTAATCTGCATCTCTAGCCAGGGTCTCATCCGCATTCGAGTGA Reference\nTAACACTCACTTTATTGATCCAGATCAGCCCTGGGATT-GGTTGGAATTAATCTGCATCTCTAGCCAGGGTCTCATCCGCATTCGAGTGA -1bp\nTAACACTCACTTTATTGATCCAGATCAGCCCTGG-----GATTGGAATTAATCTGCATCTCTAGCCAGGGTCTCATCCGCATTCGAGTGA -5bp\nSTING-/-\nCACTCTGAAGTACCTAGCACTTCACCTAGCCTCGCACGA-ACTTGGACTACTGTTGAAAAACCTCTGCTGTCTGGCTGAAGAGCTGTGCC Reference\nCACTCTGAAGTACCTAGCACTTCACCTAGCCTCGCACGAGACTTGGACTACTGTTGAAAAACCTCTGCTGTCTGGCTGAAGAGCTGTGCC +1bp\nMYD88-/-\nTGGCAATGGACCAGACACAGGTGCCCGGCAGGACGTCACGGTCGGACACACACAACTTAAGCCGATAGTCTGTCTGTTCTAGTTGCCGGA Reference\nTGGCAATGGACCAGACACAGGTGCCCGG--------------------------------GCCGATAGTCTGTCTGTTCTAGTTGCCGGA -32\nTGGCAATGGACCAGACACAGGTGCCCGGCAGGACGTCA-GGTCGGACACACACAACTTAAGCCGATAGTCTGTCTGTTCTAGTTGCCGGA -1\nTRIF-/-\nGGGAGGAAGCAGAGGACAGAGGGGAAGGAGGCAGCTTAGGGGAGGCTTGGAGGGATGGTGGCTGGGCAGGAGGGCTGGATGACGTGGTGT Reference\nGGGAGGAAGCAGAGGACAGAGGGGAAGGAGGCAGC-----GGAGGCTTGGAGGGATGGTGGCTGGGCAGGAGGGCTGGATGACGTGGTGT -5\n30\n3) Validation of gene deletion: To confirm successful knockout of the target genes,\nwe performed immunoblotting (Western blot) for each edited gene product,\nincluding CASP1, CASP3, CASP6, CASP7, CASP9, Gsdmd, Gsdme, Mlkl, Ripk3,\nCasp8, cGas, Sting, and Irf1. The absence of protein expression in the\ncorresponding knockout cell lines verified effective gene deletion. Representative\nvalidation data are now included in New Data, Extended Data Fig. 3a, 3b, 4e [shown\nbelow].\nWe believe that the inclusion of this detailed information enhances the transparency and\nreproducibility of our study. All related datasets and validation data have been\nincorporated into the revised manuscript and supplementary materials as noted above.\nCASP1\nCASP3\nCASP6\nCASP9\nActin\n–/–1psaC\niBMDMs(Mock)\n–/–3psaC –/–6psaC –/–7psaC –/–9psaC\nCASP7\nTW\n–/–dmdsG –/–emdsG\n–/–lklM\n–/–3kpiR\n–/–8psaC–/–3kpiR\niBMDMs(Mock)\nGSDMD\nGSDME\nMLKL\nRIPK3\nCASP8\nActin\nkcoM\nTW TW\n–/–SAGc\n–/–GNITS\n–/–1FRI –/–2miA\nHSV-1 HF\ncGAS\nSTING\nIRF1\nActin\nComment 3: The infection and transfection conditions in the experiments shown in Figs. 1 to 3\nshould be provided in more detail. What multiplicity of infection was used? When were the\nsamples harvested? When were the BMDMs transfected with the VP22-expression plasmid?\nWe appreciate your request for additional methodological clarity. In the revised manuscript,\nwe have updated the Methods section and figure legends for Figures 1–3 to include the\nfollowing detailed information:\n1) Multiplicity of infection (MOI): For all HSV-1 infection and viral DNA transfection\nexperiments involving primary murine BMDMs, we used an MOI of 10 at 16 hours\npost-infection, unless otherwise noted. For THP-1–derived human macrophages,\nan MOI of 5 at 16 hour post infection was used to achieve comparable infection\nefficiency without excessive cytotoxicity.\n2) Sample collection timing: In infection-based experiments shown in Figures 1–3,\ncells were harvested at 16 hours post-infection for immunoblotting, cytokine\nanalysis, and ASC speck quantification.\n3) VP22 expression plasmid transfection: For experiments involving VP22\noverexpression (Fig. 3), BMDMs were transfected with the VP22 expression\nplasmid 12 hours prior to viral infection using DOTAP (Roche, 11202375001). Cells\nwere then infected with HSV-1 and harvested 16 hours post-infection.\nThese clarified parameters are now explicitly described in the updated Methods section to\nensure full experimental reproducibility.\nComment 4: In Extended Data Fig. 3a, the numbers (1)–(3) should be (6)–(8)?\nThank you for identifying this labeling error. We have carefully reviewed Extended Data\nFig. 3a and confirm that the numbers (1)–(3) were indeed incorrect. As you correctly noted,\nthese should be labeled as (6)–(8) to accurately reflect the experimental conditions.\nThis has been corrected in the revised figure and figure legend. We appreciate your\nattention to detail, and this change has helped improve the clarity and accuracy of the\nmanuscript.\nIn the present work Oh et al study the inflammasome activation by Herpes Simplex Virus 1 strains\nin Mice and BMDMs and identify a poly(T) sequence of the HF strain that specifically activate\nAim2. Although it is well established that dsDNA induce Aim2 activation, and that the Aim2 protein\nper se could have relatively broad specificity for different dsDNA, this study emphasis that in the\ncellular context the situation is very much more controlled and that (as far as I can see) the poly(T)\nsequence constitutes the first HSV1 sequence identified that can drive activation of innate\nimmunity through Aim2. Due to this, and when our understanding of the role of Aim2 in virus\nimmunity is still quite fragmented, I feel this work could be well suited for publication in Nature\ncommunication.\nOverall, the study comprise quite comprehensive data and although the methodology used\n(knockdowns, IP, etc) is often open for multiple possible interpretations of what is going on in the\ncellular context, it appears as the data support most of their conclusions. However, I have few\nspecific concerns that I feel should be addressed:\nWe sincerely thank you for taking the time to provide constructive comments and\ninvaluable suggestions, which have significantly improved the clarity and depth of our\nmanuscript. We are particularly grateful for your encouraging remarks that “this work\ncould be well suited for publication in Nature Communications” and that “the data support\nmost of their conclusions.” These comments are highly motivating and affirm the direction\nof our study.\nIn response to your thoughtful feedback, we have further elaborated on the underlying\nmechanisms and conducted additional experiments to strengthen the evidence supporting\nour conclusions. We believe that these new data provide multiple, independent lines of\nsupport for our central findings. Below, we address each of your specific comments in\ndetail.\n- Several readout assays for Aim2 induction are used through the study, mainly Il1b/il18 release,\nCasp1 cleavage and cell death which in most cases appears to support a textbook view of Aim2\nactivation and signaling. However, the knockdown experiments shown in Extended Data Fig 2 of\nproteins that could be involved downstream of Aim2 activation is poorly followed up and the\napparent observation that cell death is not dependent on Casp1 or GSDMD/E is intriguing and\nwould contradict the textbook view. In a HSV1 infection (and the transfection of full virus DNA) it\nis quite possible that multiple pathways are activated and, although it is clear that Aim2 signaling\nis one of these, there might be other pathways contributing to e.g. to cell death and Il1b/Il18\nrelease in parallel (Yes, the Aim2 KD support that Aim2 is a key player, but these are KD clones\nwith all the possible caveats, where compensatory and complex distortions of immune signaling\nmight have been induced due by KD of Aim2). The Casp1 or GSDMD KD data is confusing and\nit is also possible that these knockdown experiments have not worked well/are not informative\n(where are the WBs showing that these proteins are indeed knocked down?). I feel the authors\nshould add some further follow-up experiment on at least the Casp1 knockdown to show whether\ndiscussions are very sparse and the work would benefit from the authors further develop there\npoint on e.g. RIPK3 dependence in the discussion and potentially also add clarifying experiments.\nCan it be excluded that there is a significant Toll receptor effect contributing to Il1b/Il18\nrelease or apoptosis? Etc\nThank you for this thoughtful and insightful comment, which has prompted us to further\nclarify the mechanistic framework of our study. In the revised manuscript, we have\naddressed each of these concerns through new experimental data and an updated\ndiscussion.\nFirst, we have confirmed the successful knockout of the following genes using\nimmunoblotting at the protein level: Casp1, Casp3, Casp6, Casp7, Casp9, Gsdmd, Gsdme,\nMlkl, Ripk3, and Casp8. All gene-edited cell lines displayed complete or near-complete loss\nof their respective proteins, as shown in New Data, Extended Data Fig. 3a, 3b, confirming\nthat the observed phenotypes are not due to partial knockdown or incomplete editing.\nCASP1\nCASP3\nCASP6\nCASP9\nActin\nAs expected, CASP1 knockout BMDMs showed a substantial reduction in IL-1β and IL-18\nsecretion following HSV-1 infection (New Data, Extended Data Fig. 4a, 4b), confirming the\nessential role of caspase-1 in cytokine maturation. Interestingly, cell death was still\nobserved in CASP1-deficient cells, suggesting that additional caspase-1–independent cell\ndeath pathways are also engaged during HSV-1 infection.\n–/–1psaC\niBMDMs(Mock)\n–/–3psaC –/–6psaC –/–7psaC –/–9psaC\nCASP7\nTW\n–/–dmdsG –/–emdsG\n–/–lklM\n–/–3kpiR\n–/–8psaC–/–3kpiR\niBMDMs(Mock)\nGSDMD\nGSDME\nMLKL\nRIPK3\nCASP8\nActin\nkcoM\nTW TW\n–/–SAGc –/–GNITS\n–/–1FRI –/–2miA\nHSV-1 HF\ncGAS\nSTING\nIRF1\nActin\n300\n240\n180\n120\nHF\nTo better understand these non-canonical mechanisms, we referred to our previous study\n(Lee et al., Nature 2021 [PMID: 34471287]), in which we demonstrated that AIM2 can\ncoordinate with other innate immune sensors such as Pyrin and ZBP1 to form a\nmultiprotein complex known as the PANoptosome. This platform mediates PANoptosis, a\nform of inflammatory cell death that integrates pyroptosis, apoptosis, and necroptosis. In\nthat work, we showed that AIM2 drives the assembly of a complex containing ASC,\ncaspase-1, RIPK3, caspase-8, and FADD, leading to inflammatory cell death during viral\ninfection.\nBuilding on this concept, our previous study demonstrated that RIPK3 plays a critical role\nin the assembly and function of the AIM2-driven PANoptosome. Specifically, in Ripk3\nknockout BMDMs infected with HSV-1, we observed a marked reduction in ASC–caspase-\n8 colocalization and impaired cell death, indicating that RIPK3 is essential for full\nexecution of the PANoptotic cell death program.\nTo address the possibility that Toll-like receptor (TLR) signaling contributes to cell death,\nwe infected MYD88 KO and TRIF KO iBMDMs with HSV-1 HF. No significant changes in cell\ndeath were observed compared with that of WT iBMDMs, indicating that TLR pathways are\nnot major contributors to the inflammatory cell death observed in our model (New Data,\nExtended Data Fig. 4c, 4d).\n–/–1psaC\n60\n)lm/gp(β1-LI\n****\nHSV-1\nHF\nTW\n–/–1psaC\n1000\n800\n600\n400\n200\n0\n)lm/gp(\n81-LI\na b\n****\n100\n75\n50\n25\n0\nhtaed\nllec\n%\n)sllec+xotyS(\nHSV1 HF\nTW\n–/–88dyM\nns\nFH\n1VSH\n–/–firT\nWT Myd88–/– Trif–/–\nTogether, these findings support a model in which AIM2 recognizes viral DNA and initiates\nPANoptosis via a multi-sensor inflammasome complex involving RIPK3 and caspase-8, in\naddition to caspase-1, and that TLR signaling does not play a dominant role in this process.\nWe have revised the Results section accordingly to incorporate this expanded mechanistic\nunderstanding.\n- The section addressing a potential role of VP22 is an inhibitor of Aim2 is not very crisp and the\nexperiments might eventually not be informative. The initial paragraph contains a somewhat\nsuperficial discussion of structural regions and what could be functionally significant. In the end\nof a paragraph, they say “Variants outside of this core domain are thus not expected to\nsignificantly impact VP22 functions and are consequently categorized as functionally neutral”.\nAlthough e.g. Aints et al addresses other types of functions, I cannot really find the data excluding\nthat other regions could have additional functions, e.g. in the context of Aim2 inhibition. Although\nthese regions might be intrinsically disordered, they could make important interactions. This\nparagraph should be rewritten, e.g. to instead just describe what is known about the structure (if\nthe authors do not know about more crisp data excluding functional significance of specific\nregions).\nThank you for this valuable comment and agree that the functional interpretation of VP22’s\nstructural domains warrants a more cautious and precise discussion.\nTo directly investigate whether VP22 suppresses AIM2 inflammasome activation in the HF\nstrain, we generated a VP22-null mutant virus in the HF background using CRISPR/Cas9-\nmediated genome editing (New Data, Extended Data Figure 5a, 5b [shown below]).\n36\na HSV-1 genome\nU U\nL S\nTRl VP22 IRl IRs TRs\ngRNA\nViral DNA VP22\neGFPdonor\nHAS2 eGFP CMVp HAS1\ntemplate\nPoly A\nb\nF1 F2\nWT VP22\nR1 R2\nF1 F2\nΔVP22 eGFP CMVp\nPoly A R1 R2\nHF F\n2 2 2 2\n2 2 2 2\nP P P P\nTV TV TV TV\nWΔ WΔ WΔ WΔ\n2 kb - 2 kb -\n1 kb -\n1 kb -\nF1+R1 F2+R2 F1+R1 F2+R2\nWe then infected primary BMDMs with this mutant virus and assessed caspase-1\nactivation, and IL-1β and IL-18 secretion as key readouts of inflammasome activation. The\nresults showed that VP22 deletion in the HF strain did not alter inflammasome activation,\nwith responses comparable to those induced by WT HF virus (New Data, Fig. 3h–3j). These\nfindings suggest that, in the HF background, VP22 is not required for the suppression or\nactivation of the AIM2 inflammasome. Accordingly, the enhanced AIM2 activation observed\nduring HF infection is most parsimoniously explained by HF-specific genomic features,\nsuch as the long poly(T) DNA sequence.\n37\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\nHF HF\nWT DVP22\nActin\n)lm/gp(\nβ1-LI\n**** 120\n90\n60\n30\nWT\nTW\n–/–2miA\nns\n150 ****\nTW\n–/–2miA\nHF\nDVP22\n–/–2miA\n)lm/gn(\n81-LI\n2.0 ****\n1.5\n1.0\n0.5\nWT\nTW\n–/–2miA\nns\n****\nTW\n–/–2miA\nHF\nDVP22\nIn contrast, a distinct outcome was observed in the F strain background. While WT F virus\nfailed to induce detectable caspase-1 activation or IL-1β and IL-18 secretion, infection with\nthe VP22-null F mutant robustly induced these inflammasome-associated readouts (New\nData, Extended Data Figure 5e, 5f, 5g [shown below]). These findings demonstrate that,\nunlike HF, AIM2 inflammasome activation in the F strain is VP22-dependent. This result is\nconsistent with previous reports showing that VP22 suppresses inflammasome activation\nin HSV-1 F and related strains (PMID: 29447697).\nIn light of the reviewer’s suggestion, we have revised the paragraph discussing VP22 to\nmake it more concise and evidence based. Rather than inferring functional neutrality of\nsequence variations outside the conserved core domain, we now limit the description to\nestablished structural features of VP22, including the presence of a conserved core region\ndefined by prior structural and comparative studies.\nWe also acknowledge that regions outside the structured core are less well-characterized,\nand therefore have refrained from making definitive statements regarding their functional\nrelevance, particularly in the context of AIM2 inflammasome regulation. This revision\navoids overinterpretation and more accurately reflects the current understanding and\nlimitations of VP22 structure–function relationships.\nWe appreciate your constructive critique, which has helped us refine both the experimental\nand conceptual clarity of this section.\n- It is concluded from the VP22 over-expression experiment that there is no differential effect of\nVP22 for the different strains. However, there is no positive control experiment showing that the\nVP22 overexpression in this system can inhibit Aim2. Therefore, I would like to see a control\nexperiment demonstrating that VP22 overexpression can indeed inhibit Aim2 activation, e.g. with\na highly potent dsDNA for Aim2 activation. Otherwise, there is no use addressing differential effects.\n38\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\ne F F\nWT DVP22\nActin\n)lm/gp(\nβ1-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n200 ****\nTW\n–/–2miA\nns\nF\nDVP22\n)lm/gp(\n81-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n****\n–/–2miA\nf g\n250\nns\nF\nDVP22\nThank you for this important and constructive suggestion. To determine whether VP22 can\ninhibit AIM2 inflammasome activation, we performed a set of control experiments using\npotent synthetic AIM2 ligands, including poly(dA:dT) and poly(dG:dC), as well as dsDNA\npoly(T) 90-mer, which was shown in this study to robustly activate AIM2 (New Data,\nExtended Data Figure 6e-6h [shown below]). In BMDMs transfected with any of these\ndsDNA ligands, we consistently observed strong caspase-1 activation and IL-1β secretion,\nconfirming effective AIM2 inflammasome engagement.\nWe next performed immunoblotting to confirm VP22 expression following transfection of\nthe VP22 expression vector. In mock-infected BMDMs, VP22 was expressed at detectable\nlevels upon transfection, confirming successful overexpression. Moreover, subsequent\ninfection with HSV-1 HF did not further enhance VP22 expression. Importantly, VP22\nexpression levels were comparable between WT and Aim2−/− BMDMs under all conditions\ntested (New Data, Figure 3b [shown below]). These results indicate that VP22 expression\nin primary macrophages is not readily augmented by exogenous overexpression or viral\ninfection and is not influenced by AIM2 deficiency. Importantly, even under these\nphysiologically relevant conditions, HF infection robustly activated the AIM2\n39\n1PSAC\nTW\n–/–2miA\nP45\nP20\nActin\nTW\n–/–2miA\nTW\n–/–2miA\nT) C)\nA: d G: d\nM\nock\nP\noly(d\nP\noly(d\n200\n160\n120\n80\n–/–2miA\nTW\n–/–2miA\n40\n)lm/gp(\nβ1-LI\n****\n****\nT) C)\nA: d G: d\nP\noly(d\nP\noly(d\n10 30 90\n1PSAC\nWT Aim2–/–\n****\nC 10 30 90 C mer 400 WT\nP45 300 Aim2–/–\n200\n100 ns ns ns\nP20\nActin 0\n)lm/gp(\nβ1-LI\nControl 10 mer 30 mer 90 mer\ninflammasome, supporting our conclusion that VP22 does not inhibit HF-induced AIM2\ninflammasome activation.\nb\nVP22\nActin\nTo further investigate the role of VP22 in AIM2 inflammasome regulation, we generated\nVP22-null mutant viruses in both the HF and F strain backgrounds using CRISPR/Cas9-\nmediated genome editing. Successful deletion of VP22 was confirmed by immunoblot\nanalysis, which showed complete loss of VP22 protein expression in the mutant viruses.\nComparison of viral protein expression between WT and VP22-null viruses revealed that\noverall viral protein levels were slightly reduced in the VP22-null mutants compared with\nWT viruses, consistent with the known role of VP22 in optimal viral fitness (New Data,\nExtended Data Figure 5c, 5d [shown below]).\nIn the HF background, BMDMs infected with WT HF or VP22-null HF showed comparable\nlevels of caspase-1 cleavage and IL-1β and IL-18 secretion (New Data, Figure 3h, 3i, 3j\n[shown below]). These results indicate that deletion of VP22 does not alter AIM2\ninflammasome activation in the HF strain, supporting our conclusion that VP22 is not a\ncritical regulator of inflammasome activation in this context. Accordingly, the enhanced\nAIM2 activation observed during HF infection is most parsimoniously explained by HF-\nspecific genomic features, such as the long poly(T) DNA sequence.\nMedia\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\nControl\n–/–2miA\nVP22\nHF Media HF\nHSV-1 HF HSV-1 F\nc d\nWT ∆VP22 WT ΔVP22\nM 3 6 9 3 6 9 (hpi) M 3 6 9 3 6 9 (hpi)\nICP0 ICP0\nICP8 ICP8\nVP22 VP22\ngC\ngC\nGAPDH\nGAPDH\nIn contrast, a distinct outcome was observed in the F strain background. While WT F virus\nfailed to induce detectable caspase-1 activation or IL-1β and IL-18 secretion, infection with\nthe VP22-null F mutant robustly induced these inflammasome-associated readouts (New\nData, Extended Data Figure 5e. 5f. 5g [shown below]). These findings demonstrate that,\nunlike HF, AIM2 inflammasome activation in the F strain is VP22-dependent. This result is\nconsistent with previous reports showing that VP22 suppresses inflammasome activation\nin HSV-1 F and related strains (PMID: 29447697).\nTaken together, our data reveal a strain-dependent role of VP22 in regulating AIM2\ninflammasome activation. While VP22 functions as an inhibitory factor in F strain infection,\nit is dispensable for inflammasome regulation in the HF strain, where viral DNA features\nsuch as poly(T) DNA play a dominant role. These new data have been included in the\nrevised manuscript in the Results section.\n41\n)lm/gp(\nβ1-LI\n****\n120\n90\n60\n30\nWT\nTW\n–/–2miA\nns\n150\n****\nTW\n–/–2miA\nHF\nDVP22\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\nHF HF\nWT DVP22\nActin\n–/–2miA\n)lm/gn(\n81-LI\n2.0 ****\n1.5\n1.0\n0.5\nWT\nTW\n–/–2miA\nns\n****\nTW\n–/–2miA\nh i j\nHF\nDVP22\n1PSAC\nMock\nP45\nP20\nTW\n–/–2miA\nTW\n–/–2miA\nTW\n–/–2miA\ne F F\nWT DVP22\nActin\n)lm/gp(\nβ1-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n200 ****\nTW\n–/–2miA\nns\nF\nDVP22\n)lm/gp(\n81-LI\n150\n100\n50\nWT\nTW\n–/–2miA\nns\n****\n–/–2miA\nf g\n250\nns\nF\nDVP22\n- In the IP figures (e.g. Fig2 F), in the Input chart, there is no Aim2 protein present for the WT\nmedia lane. This suggests that the virus first induce Aim2, which is subsequently activated by\nviral dsDNA (?). How is Aim2 induced in this context? It would be particuliarly interesting to shed\nlight on how this is accomplished for HF seq#5 when this is a requirement for Aim2 activation.\nThank you for raising this important question regarding the regulation of AIM2 expression\nin response to viral infection.\nIndeed, as you observed, AIM2 protein was not detectable in uninfected BMDMs (WT media\nlane). This observation is consistent with previous studies (e.g., PMID: 34471287, 38008850,\n41225161), which have shown that AIM2 is not constitutively expressed at high levels\nunder resting conditions, and is instead inducible upon innate immune stimulation.\nThe upstream regulatory mechanisms controlling AIM2 expression have been well\ndescribed in the literature. Notably, prior studies (e.g., PMID: 25774715, 37557956) have\ndemonstrated that AIM2 transcription is directly regulated by IRF1, a transcription factor\nactivated downstream of various cytosolic DNA sensing pathways, including cGAS–STING\nsignaling.\nTo investigate whether this pathway contributes to AIM2 induction in our system, we\ngenerated cGAS, STING, and IRF1 knockout (KO) iBMDMs and examined cell death\nresponses in IRF1 KO, cGAS KO, and STING KO iBMDMs following HSV-1 HF infection.\nInterestingly, all three KO lines exhibited marked suppression of virus-induced cell death,\ncomparable to the phenotype observed in AIM2 KO cells (New Data, Extended Data Figure\n4f, 4g [shown below]). These findings support a model in which the cGAS–STING–IRF1\naxis mediates the transcriptional induction of AIM2, enabling subsequent inflammasome\nactivation by viral DNA.\nWe have included these mechanistic insights and supporting experimental data in the\nrevised manuscript to clarify how AIM2 inflammasome is regulated in the context of HSV-\n–/–saGc –/–gnitS\n–/–1frI\n–/–2miA\n100\n75\n50\n25\n0\nhtaed\nllec\n%\n)sllec\n+xotyS(\n****\nWT cGas–/– Sting–/– Irf1–/– Aim2–/–\nHSV1 HF\nFH\n1VSH\nf g\n- I feel the Discussion section is quite thin, and in addition to above points on the lack of discussion\nof mechanistic implications, it should be possible to further address the implications of the poly(T)\nsequence for virus immune response. Are there other Herpesviruses, other viruses, containing\nsimilar sequences. This sequence is relative short in relationship to\nsome other Aim2 inducing sequences, etc\nThank you for this thoughtful suggestion, which has prompted us to more thoroughly\nexplore the immunological significance of the poly(T) sequence and its evolutionary\ncontext across viruses.\nFirst, we examined 492 complete HSV-1 genome datasets from the NCBI database. Among\nthese, 18 sequences contained a 23-mer poly(T) sequence, including several well-\ncharacterized laboratory strains (e.g., H166, KOS63, ZW63) and multiple clinical isolates\n(e.g., from Greninger, A.L.; Rathbun, M.; Furlong, J.; Johnston, C.). These data indicate\nthat the 23-mer poly(T) sequence is not unique to HF or lab-adapted strains but is also\npresent in naturally circulating viral populations (New Data, Extended Data Figure 10a\n[shown below]).\n80\n75.0%\n70\n60 61.1% Clinical sample\nKOS63\n30 27.7% Others\n20\n13.1%\n10 8.3%\n5.5% 5.5%\n2.3% 1.1%\n23 mer 14 mer\nAdditionally, we found that a 14-mer poly(T) sequence, which we used as a reference for\nthe ΔT mutant, was present in approximately 15% (63/426) of clinical HSV-1 samples,\n14\nwhile the 23-mer poly(T) sequence appeared in 3% (11/426) of those clinical datasets (New\nData, Extended Data Figure 10b [shown below]). This pattern of distribution supports the\ninterpretation that poly(T) sequence length variability is a naturally occurring\npolymorphism among HSV-1 strains, rather than a cell culture–induced artifact such as\nthose seen in syncytial mutations.\n43\negatnecreP\n%\na\nb\n100\n80\n60\n40\n20\n0\not\nal\nS a m\npl e\nH 1\n6 6\nK O S\n6 3\nR\nH 2\nni c\nal\ns a m\npl e\nOt h\ner s\nT Cli\nTo further explore the biological relevance of these poly(T) tracts, we conducted a broader\ncross-species sequence analysis using the NCBI viral genome repository. Among the DNA\nviral genome datasets containing a 23-mer poly(T) sequence, we found that about 90% are\nfrom Monkeypox virus, Human herpesvirus and adenovirus. Among the RNA viral genome\ndatasets containing a 23-mer poly(T) sequence, we found that about 90% are from\nHepacivirus and SARS-Coronavirus (New Data, Supplementary Table 1).\nFor the 14-mer poly(T) motif, in addition to Monkeypox virus, we detected matches in\nHuman herpesvirus 4 (EBV), Human papillomavirus 16 (HPV16), and Human\nmastadenovirus D (HAdV-D) among DNA viruses. Among RNA viruses, we identified\npoly(T)-containing sequences in Hepacivirus, SARS-CoV-2, and Classical Swine Fever\nVirus (CSFV).\nThese findings indicate that long poly(T) tracts are not exclusive to HSV-1, but rather are\nconserved across a broad spectrum of clinically important DNA and RNA viruses. This\nwidespread occurrence suggests that poly(T) sequences may play a previously\nunderappreciated role in innate immune sensing, and further supports the relevance of\nour findings in a broader virological and immunological context.\nIn conclusion, both intra-species HSV-1 analysis and cross-species comparisons strongly\nrefute the idea that the 23-mer poly(T) sequence is a culture-derived artifact. Instead, the\ndata support the view that this sequence represents a naturally occurring, functionally\nrelevant genetic motif that may shape strain-specific host responses via AIM2\ninflammasome activation.\nAccording to the reviewer’s comment against the thin Discussion section, we added the\nfollowing paragraph at the end of the Discussion:\n44\nnoitroporP\n%\n100% 100%100%\n78%\nPoly(T) 14 mer\nPoly(T) 23 mer\n56%\n50%\n20%\n17% 15%\n4% 3% 0%\nFuture studies will be required to determine how poly(T) length polymorphisms influence\ndisease severity in patients and whether similar DNA motifs contribute to inflammasome\nactivation in other viral or inflammatory disorders. Systematic profiling of poly(T)-rich\ngenomic regions across viral genomes, coupled with clinical outcome data, may further\nelucidate how viral genomic variation shapes innate immune responses in vivo.\nCollectively, our study establishes viral poly(T) DNA as a physiologically relevant ligand\nthat governs strain-specific AIM2 inflammasome activation during HSV-1 infection. By\nuncovering how naturally occurring genomic sequence variation shapes innate immune\nsensing, these findings advance fundamental understanding of host-virus interactions\nand highlight poly(T) DNA as a potential target for therapeutic modulation of\ninflammasome responses. More broadly, this work provides a conceptual framework for\nexploring how subtle viral genome polymorphisms can decisively influence immune\noutcomes, extending beyond HSV-1 to other viral and inflammatory diseases.\n45"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised manuscript presents compelling genetic and functional evidence that viral poly(T)\nDNA length polymorphisms determine strain-specific AIM2 inflammasome activation during HSV-\n1 infection. The inclusion of precise deletion, reversion, and insertion mutants, together with in\nvivo validation and confirmation in human macrophages, substantially strengthens the\nmechanistic framework and causal inference.\nThe study is carefully designed, technically rigorous, and conceptually significant. I have only\nminor suggestions aimed at further improving clarity and transparency:\nClarification of DNA length context\nThe manuscript demonstrates that efficient AIM2 activation requires dsDNA above a length\nthreshold (~≥80 bp), while the HF strain contains a 27-mer poly(T) tract embedded within genomic\nDNA. A brief clarification in the Discussion explaining how the genomic context provides sufficient\nDNA length for AIM2 oligomerization would further strengthen mechanistic clarity.\nStatistical reporting consistency\nPlease ensure that exact sample sizes (n), number of biological replicates, and statistical tests\nare consistently reported in all figure legends, including Extended Data figures.\nImmunoblot presentation\nWhere feasible, include molecular weight markers or indicate expected molecular weights in\nfigure legends for key immunoblots to enhance transparency.\nThese are presentation-level refinements and do not affect the central conclusions.\nOverall, this is a strong and well-supported study that provides important insights into viral\ngenome–encoded determinants of innate immune sensing.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have performed an extensive and impressive set of additional experiments, and the\nrevised manuscript is substantially strengthened compared to the original submission.\nI am largely satisfied with the authors’ responses and the revised manuscript, and I support its\npublication in Nature Communications after minor revision. I have two suggestions that would\nfurther improve the clarity and presentation of the work.\nFirst, in the revised Fig. 3b, the authors have added Western blotting data to confirm VP22\nexpression following transient overexpression. While this demonstrates that VP22 protein is\nproduced, western blotting has inherent limitations in this experimental context, as it cannot verify\nwhether the VP22-expressing cells are identical to the productively infected cells. Given that the\nauthors have now successfully generated and analyzed genetically defined VP22-deficient\nviruses in both the HF and F strain backgrounds, the transient VP22 overexpression data (Fig.\n3b–e) are of reduced central importance. In contrast, the ΔVP22 virus data provide more direct\nand physiologically relevant evidence regarding the strain-dependent requirement of VP22.\nI therefore suggest moving the current Fig. 3b–e to the Extended Data section. Conversely, since\none of the key conclusions of this study is the differential role of VP22 in HF versus F strains,\nthe data for the F strain ΔVP22 mutant (currently shown in Extended Data Fig. 5e–g) would be\nmore impactful if included in the main Figure 3. This rearrangement would facilitate direct\ncomparison between strains and further clarify the distinct roles of VP22 in each viral context.\nSecond, although the authors’ large-scale genomic analyses strongly support the presence of\nlong poly(T) tracts in both laboratory strains and clinical isolates, many HSV-1 genome sequences\ndeposited in public databases have undergone cell-culture passage. As such, statements\ndescribing these long poly(T) motifs as strictly “naturally occurring” may be somewhat overstated.\nWhile the data are clearly consistent with their presence in circulating viral populations, it remains\ndifficult to completely exclude a contribution from cell-culture adaptation. I therefore recommend\nslightly softening the wording in the relevant sections (e.g., pages 14–15 of the revised\nmanuscript), using phrasing such as “consistent with naturally occurring variation” or “present in\nboth clinical and laboratory isolates,” to avoid over-interpretation without diminishing the impact\nof the findings.\nWith these minor revisions, I believe the manuscript will be suitable for publication."}, "author_response": "We sincerely thank the reviewer for the positive and thoughtful evaluation of our work. We greatly appreciate the reviewer’s recognition of the conceptual significance of our study and the rigorous experimental design supporting our conclusions. We are also grateful for the helpful suggestions aimed at improving the clarity and transparency of the manuscript.\nWe have carefully considered these comments and revised the manuscript accordingly.\n\nClarification of DNA length context\nWe thank the reviewer for this insightful suggestion. We agree that clarifying the relationship between the poly(T) motif and the DNA length requirement for AIM2 activation improves the mechanistic clarity of our study. Although the poly(T) tract itself is 27 nucleotides in length, it is embedded within the much longer HSV-1 genomic DNA, which provides an extended dsDNA platform capable of supporting AIM2 filament formation and inflammasome assembly. Thus, the poly(T) motif likely functions as a sequence determinant within a longer genomic DNA context rather than as an isolated ligand. We have now added a brief clarification in the Discussion to explain this point.\n\nStatistical reporting consistency\nWe thank the reviewer for this helpful suggestion. We have carefully reviewed all figure legends and ensured that the exact sample sizes (n), number of biological replicates, and statistical tests are consistently reported throughout the manuscript. This information has now been clearly indicated in all figure legends, including those of the Extended Data figures.\n\nImmunoblot presentation\nWe sincerely thank the reviewer for the positive and encouraging evaluation of our study. We greatly appreciate the reviewer’s recognition of the significance of our findings and the strength of the experimental evidence supporting our conclusions.\nIn response to this suggestion, we have revised the figure legends to indicate the expected molecular weights for key immunoblot bands where appropriate. These additions improve the transparency and clarity of data presentation.\nWe thank the reviewer for these helpful suggestions, which have further improved the presentation of the manuscript.\n\nReviewer #2 (Remarks to the Author):\nWe appreciate your participation in the co-review process and the Nature Communications initiative to support Early Career Researchers. No further comments were provided in this section, so we thank you for your support.\n\nReviewer #3 (Remarks to the Author):\nWe sincerely thank the reviewer for the positive and encouraging evaluation of our revised manuscript. We greatly appreciate the reviewer’s recognition that the additional experiments have strengthened the study and improved the manuscript. We are also grateful for the supportive comments regarding publication in Nature Communications.\nWe have carefully considered the reviewer’s suggestions and revised the manuscript accordingly to further improve the clarity and presentation of the work.\n\nFirst, we thank the reviewer for this insightful suggestion regarding the presentation of the VP22-related data. We agree that the genetically defined ΔVP22 virus experiments provide more direct and physiologically relevant evidence for the strain-dependent role of VP22 during HSV-1 infection.\nFollowing the reviewer’s suggestion, we have reorganized the figures accordingly. The transient VP22 overexpression data (previously shown in Fig. 3b–e) have been moved to the Extended Data section, as they primarily serve as supportive validation of VP22 expression. Conversely, the data for the F strain ΔVP22 mutant (previously shown in Extended Data Fig. 5e–g) have now been incorporated into the main Figure 3, enabling clearer comparison between HF and F strain backgrounds.\nWe agree that this rearrangement improves the clarity of the presentation and more directly highlights the distinct roles of VP22 in the two viral contexts.\n\nSecond, we thank the reviewer for this thoughtful suggestion. We agree that, although our genomic analyses support the presence of long poly(T) tracts in both laboratory strains and publicly available HSV-1 genomes, it is difficult to completely exclude the potential contribution of cell-culture passage in some database sequences.\nFollowing the reviewer’s recommendation, we have softened the wording in the relevant sections of the manuscript (pages 14–15). Specifically, we have revised the text to use more cautious phrasing such as “present in both clinical and laboratory isolates” and “consistent with naturally occurring variation,” rather than describing these motifs as strictly “naturally occurring.”\nWe appreciate this suggestion, which has helped us improve the accuracy and clarity of our interpretation without altering the overall conclusions of the study."}]

Decision letter