Hydroxylation of the HIV-1 antisense protein promotes immune evasion of HIV-1 via modulation of TBK1

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

Abstract
HIV-1 evades host immunity via a number of virally encoded mediators. Here we show that the HIV-1 antisense protein (ASP) evades host immunity by interfering with the type I interferon (IFN-I) response. Mechanistically, host prolyl hydroxylase 3 (PHD3) hydroxylates ASP at Pro47, enabling the recruitment of the RING finger protein 114 (RNF114) to TANK-binding kinase 1 (TBK1). Subsequently, RNF114 mediates K6-linked ubiquitination of TBK1 at Lys236, suppressing TBK1 activation and the downstream IFN-I response. Conversely, mutation of ASP at Pro47 abolishes this inhibitory effect. In humanized mice, either ASP deletion or treatment with the RNF114 inhibitor EN219 or the PHD3 inhibitor Molidustat enhances antiviral immunity and reduces viral replication. Clinically, RNF114 and PHD3 transcript levels exhibit a positive correlation with viral load in treatment-naive patients. Here we show a distinct HIV-1 immune evasion mechanism involving proline hydroxylation and K6-linked ubiquitination, highlighting therapeutic potential.

Introduction
HIV is a retrovirus that targets the host immune system, ultimately leading to acquired immunodeficiency syndrome (AIDS)1. Both the DNA and RNA forms of HIV-1 are detected by cytosolic and endosomal pattern recognition receptors (PRRs), including RIG-I, MDA5, DDX3, cyclic GMP-AMP synthase (cGAS), IFI16, PQBP1, and Toll-like receptors (TLR3, TLR7, TLR8)2. Among these, RIG-I plays a pivotal role in detecting HIV-1 genomic RNA. Upon viral RNA binding, activated RIG-I engages mitochondrial antiviral signaling protein (MAVS), promoting the formation of MAVS prion-like aggregates. These aggregates recruit TNF receptor-associated factor 3 (TRAF3), which undergoes K63-linked polyubiquitination by the MAVS complex. Polyubiquitinated TRAF3 then recruits and activates TANK-binding kinase 1 (TBK1), resulting in TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3) and subsequent inducing of type I interferons (IFN-I) and antiviral responses3. In parallel, Y-form HIV-1 DNA generated during reverse transcription is specifically recognized by cGAS. Upon DNA binding, cGAS synthesizes cyclic GMP–AMP (cGAMP), a secondary messenger that activates stimulator of interferon genes (STING). Activated STING recruits TBK1, leading to TBK1-dependent phosphorylation of IRF3. Phosphorylated IRF3 dimerizes, translocates to the nucleus, and induces transcription of IFN-I and pro-inflammatory cytokines4, 5.

Despite the high sophistication and efficiency of the host immune system, HIV-1 has evolved multiple mechanisms to disrupt signaling pathways and suppress interferon production, facilitating immune evasion. During acute infection or interruption of antiretroviral therapy, HIV-1 displays pronounced resistance to IFN-I, thereby undermining innate antiviral defenses6. This resistance is associated with impaired TLR3-mediated activation in microglia and macrophages, resulting in reduced expression of IFNs and key antiviral genes7. Additionally, N6-methyladenosine (m6A) modifications on HIV-1 RNA enable the virus to evade RIG-I-mediated recognition, suppress downstream activation of IRF3 and IRF7, and inhibit IFN-I induction in monocytic cells and macrophages8. HIV-1 proteins also actively target host signaling components: Vpu and Nef disrupt the adaptor protein IPS-1 to inhibit IFN-I production in T cells, dendritic cells, and macrophages9; direct interaction with the CD303 receptor reduces and delays IFN-α production in plasmacytoid dendritic cells10, and Vpr and Vif suppress IFN induction by blocking TBK1 phosphorylation11. Moreover, even in the presence of exogenous IFN-α, HIV-1 impairs the JAK-STAT pathway through Vpu- and Nef-mediated inhibition of STAT1 phosphorylation12.

The HIV-1 antisense protein (ASP) is encoded by an open reading frame on the negative strand of the viral genome, overlapping the env gene in reverse orientation13. ASP is a highly hydrophobic transmembrane protein that localized to the nucleus, plasma membrane, and viral envelope. At baseline, it resides in the nucleus with minimal suppressive epigenetic marks but translocates to the cytoplasm and cell surface upon stimulation14. ASP expression has been validated both in vitro and in vivo, supported by antibody detection in patient sera and the induction of ASP-specific CD8⁺ T-cell responses during chronic infection15. Notably, ASP is exclusively conserved in pandemic group M HIV-1 strains, where its intact gene emerged in parallel with this group and correlates with accelerated disease progression16. Despite more than three decades of research, ASP’s precise role in immune evasion remains incompletely understood and actively debated17.

In this study, we demonstrated that ASP suppresses host IFN-I production during the early stages of infection, thereby facilitating immune evasion. Mechanistically, ASP interacts with prolyl hydroxylase 3 (PHD3), which hydroxylates ASP at Pro47. This modification promotes the recruitment of the RING finger protein 114 (RNF114) to TBK1, triggering K6-linked ubiquitination of TBK1 at Lys236. This posttranslational event attenuates TBK1 activation and suppresses cytokine production. Notably, pharmacological inhibition of PHD3 with Molidustat or RNF114 with EN219 reverses ASP-mediated IFN-I suppression and reduces HIV-1 replication, highlighting these enzymes as potential therapeutic targets for HIV infection.

Results
ASP suppresses the antiviral innate immune response
To investigate the role of ASP in host immune responses, we measured asp transcript levels in PBMCs from ART-suppressed and treatment-naive individuals. As shown in (Fig. 1a), we observed substantial asp transcription in both groups. While ASP persistence under ART aligns with mechanisms of viral latency18, its robust expression in viremic patients implies an active role. Given that IFN is the primary host barrier, we hypothesized that ASP antagonizes IFN signaling to facilitate evasion. To assess whether ASP affects IFN-I production, we overexpressed GFP-ASP in L929 cells and challenged them with herpes simplex virus 1 (HSV-1). Quantitative PCR revealed that ASP overexpression significantly reduced the transcription of Ifnb, Isg15, Cxcl5, and Tnf (Fig. 1b). Correspondingly, HSV-1 replication was substantially promoted in ASP-expressing cells (Fig. 1c). Given that the IFN-I, NF-κB, and MAPK pathways are central to antiviral immune responses19, we next examined the effect of ASP on these signaling cascades. Immunoblot analysis showed that ASP overexpression strongly inhibited the phosphorylation of TBK1, IRF3, p38, p65, and ERK (Fig. 1d), thereby suppressing IFN-I production. Consistently, both immunofluorescence and nuclear–cytoplasmic fractionation demonstrated that ASP profoundly inhibited IRF3 nuclear translocation (Fig. 1e and Supplementary Fig. 1a), a key step in IFN-I production. Since ssRNA and cDNA of HIV are detected by distinct intracellular sensors initiating divergent immune signaling cascades, we next examined whether ASP broadly modulates IFN-I responses triggered by different stimuli. L929 cells stimulated with cGAMP (a STING agonist) or vesicular stomatitis virus (VSV, an RNA virus) exhibited consistently reduced IFN-I induction upon ASP overexpression, regardless of the upstream activating signal (Supplementary Fig. 1b, c). Collectively, these findings indicate that ASP functions as a general inhibitor of IFN-I production across diverse innate immune pathways.

To determine whether ASP modulates host immune responses during HIV-1 infection, we generated pseudotyped NL4.3-Δenv HIV-1 constructs—wild-type (HIV-1) and ASP-deficient (HIV-1Δasp)—in which the envelope protein was replaced with vesicular stomatitis virus glycoprotein (VSV-G). This design restricts viral replication to a single cycle and enables efficient entry into diverse cell types20, 21. To evaluate whether asp deletion affects viral transcriptional activation, TZM-bl cells were infected with either HIV-1 or HIV-1Δasp, and luciferase activity was quantified. The results showed that there was no significant difference in the viral titer (Supplementary Fig. 2a), consistent with prior findings that asp deletion does not alter HIV-1 replication dynamics22. To further define the regulatory role of ASP during HIV-1 infection and its modulation of the antiviral immune response, we performed RNA-seq on human monocyte-derived macrophages (MDMs) infected with wild-type HIV-1 or the HIV-1Δasp. Gene set enrichment analysis revealed significant enrichment of cytosolic DNA sensing and RIG-I signaling pathways in HIV-1Δasp infected cells (Fig. 1f). Kyoto Encyclopedia of Genes and Genomes analysis highlighted significant upregulation of innate immune pathways, including MAPK and NF-κB cascades (Fig. 1g). Notably, transcripts encoding antiviral cytokines (ISG15, MX2, TNF and CCL5) were markedly upregulated, whereas anti-inflammatory genes (IL10 and CXCL12) were downregulated in HIV-1Δasp-infected MDMs (Fig. 1h and Supplementary Fig. 2b), highlighting the immunomodulatory function of ASP.

Based on these findings, we further investigated the role of ASP in macrophages and T cells. HIV-1Δasp infection of human MDMs induced substantially increased phosphorylation of TBK1, IRF3, p38, p65, and ERK (Supplementary Fig. 2c ), along with enhanced transcription of IFNB, ISG15, CXCL5, CCL5, and TNF (Supplementary Fig. 2d), while viral mRNA expression was significantly suppressed in HIV-1Δasp-infected cells (Supplementary Fig. 2e). Jurkat cells infected with HIV-1Δasp similarly showed elevated IFNG and CD69 expression (Supplementary Fig. 2f), while viral mRNA expression in Jurkat cells was significantly suppressed in HIV-1Δasp-infected cells (Supplementary Fig. 2g). Further infection assays using THP-1 cells and peritoneal macrophages (PMs) demonstrated similar results: asp deletion consistently increased phosphorylation of TBK1, IRF3, p38, p65, and ERK (Supplementary Fig. 2h) and upregulated cytokine expression (Supplementary Fig. 2i-l). To better model early infection, we next infected MDMs with replication-competent full-length pNL4-3 (YU2) HIV-1 and HIV-1Δasp under BSL-3 containment. Consistently, HIV-1Δasp-infected MDMs exhibited enhanced phosphorylation of key signaling molecules (Fig. 1i), increased cytokine transcription (Fig. 1j), suppressed tat expression (Fig. 1k), and pronounced IRF3 nuclear translocation (Fig. 1l). As a result, HIV-1Δasp exhibited significantly slower replication kinetics and markedly lower viral yields compared to wild-type throughout the 9-day time course (Fig. 1m). Collectively, these findings establish ASP as an essential factor for HIV-1 immune evasion, suppressing host IFN-I responses through multiple signaling pathways.

ASP specifically interacts with TBK1
To investigate the mechanism underlying immunosuppressive function of ASP during HIV-1 infection, we co-expressed GFP-tagged ASP with key regulators of the IFN-I pathway—including RIG-I, MAVS, cGAS, STING, TBK1, and IRF3—in HEK293T cells. Co-immunoprecipitation (Co-IP) assays revealed a strong and specific interaction between ASP and TBK1 (Fig. 2a), confirmed bidirectionally by reciprocal Co-IPs (Fig. 2b, c). Functional luciferase reporter assays demonstrated that ASP potently inhibited IFN-β promoter activation induced by cGAS-STING, RIG-I, MAVS, or TBK1, but not by IRF3, indicating that ASP acts upstream of IRF3 in the IFN-I signaling cascade (Fig. 2d). To exclude potential steric hindrance from the GFP tag, we generated HA- and Flag-tagged ASP (Supplementary Fig. 3a). Consistent with GFP-ASP, both variants specifically co-localized with TBK1 in the cytoplasm (appearing as yellow puncta) (Supplementary Fig. 3b) and potently inhibited TBK1-mediated IFN-β and ISRE activation (Supplementary Fig. 3c, d). This confirms that ASP localization and function are intrinsic and tag-independent. To characterize the ASP-TBK1 interaction, we performed in silico protein–protein docking using HDOCKlite v1.1, which predicted a high-affinity binding interface. PLIP structural analysis identified multiple hydrogen bonds (blue solid lines) and five salt bridges (yellow dashed lines), supporting a direct and stable ASP-TBK1 association (Fig. 2e). To experimentally validate this predicted interface, we introduced site-directed mutations at the key TBK1 contact residues (including Glu561, Asp586, Gln565, Glu579, Asn578, and Arg574). Consistent with the docking model, co-immunoprecipitation assays revealed that these mutations significantly attenuated ASP binding, confirming the specificity of the interaction (Fig. 2e). ASP-overexpressing L929 cells infected with HSV-1 showed enhanced ASP–TBK1 interaction by Co-IP, while confocal microscopy confirmed increased colocalization of the two proteins post-infection (Fig. 2f, g). Direct binding was further verified by GST pull-down assays, where GST-ASP interacted with His-TBK1 purified from E. coli (Fig. 2h). To map the interaction domains, we generated truncation mutants of ASP based on predicted topological features and segmented TBK1 into kinase, ubiquitin-like, and C-terminal domains. Co-IP analysis revealed that all tested ASP regions contributed to binding (Fig. 2i), whereas the ubiquitin-like domain of TBK1 was necessary for interaction with ASP, consistent with the PLIP modeling (Fig. 2j). These results demonstrate that ASP directly and specifically binds TBK1, revealing a molecular basis for ASP-mediated modulation of antiviral signaling.

ASP inhibits TBK1 activation
Given the specific ASP–TBK1 interaction, we examined whether ASP impairs IFN-I production by attenuating TBK1 activation. In vitro kinase assays demonstrated that ASP significantly inhibited TBK1-mediated phosphorylation of STING, TBK1 autophosphorylation, and IRF3 phosphorylation (Fig. 3a–c). Since TBK1 orchestrates IFN-I responses by forming complexes with STING, IRF3, and adaptors such as TRAF3, we assessed the effect of ASP on these interactions. Co-IP assays revealed that ASP expression disrupted TBK1 binding to IRF3 (Fig. 3d), STING (Fig. 3e), and TRAF3 (Fig. 3f), and suppressed IRF3 dimerization (Fig. 3g). In HIV-1-infected L929 cells, GFP-ASP overexpression inhibited virus-induced TBK1 binding to STING, IRF3, and TRAF3 (Fig. 3h). Consistently, confocal microscopy revealed significantly enhanced colocalization of TBK1 with STING (Fig. 3i) and IRF3 (Fig. 3j) in cells infected with HIV-1Δasp compared to wild-type HIV-1. Pseudovirus infection of MDMs similarly revealed markedly increased TBK1–TRAF3 and TBK1–IRF3 associations following asp deletion (Fig. 3k), further supporting the role of ASP in impeding kinase complex formation. Moreover, ASP overexpression in HSV-1-infected L929 cells substantially suppressed virus-induced IRF3 dimerization (Fig. 3l), whereas HIV-1Δasp-infected THP-1 displayed enhanced IRF3 dimerization post-pseudovirus infection (Fig. 3m). Notably, TBK1 knockdown via siRNA in PMs reversed ASP-mediated suppression, as shown by reduced transcription of Ifnb, Isg15, Cxcl5, and Tnf compared to siNC controls (Fig. 3n). Collectively, these results indicate that ASP impedes IFN-I production by inhibiting TBK1 kinase activity and its interactions with STING, TRAF3, and IRF3 within the antiviral signaling complex.

Pro47 is essential for ASP to suppress TBK1 activation
To identify structural elements critical for the immunosuppressive function of ASP, we generated a series of GFP-tagged ASP truncation mutants and expressed them in L929 cells, followed by HSV-1 stimulation. Notably, truncations containing the intracellular domains (residues 1–62 and 1–84) suppressed Ifnb mRNA expression to levels comparable to full-length ASP (Fig. 4a). A recent study revealed that rare double-cysteine residues in the HIV-1 envelope protein (Env) correlate strongly with neutralization escape and breadth development23. Therefore, ASP may also retain conserved immune-regulatory motifs during HIV evolution. Comparative sequence alignment of ASP orthologs across HIV-1 clades24 identified two conserved cysteine-rich motifs (10CCC12 and 22CCC24) and a Pro-x-x-Pro-x-x-Pro motif (47PxxPxxP53) within the 1–62 region. To assess their functional relevance, we engineered ASP mutants deleting each motif individually. Dual-luciferase reporter assays revealed that deleting of either CCC motif (Δ10–12 or Δ22–24) did not affect ASP’s inhibition of IFN-β and ISRE promoter activity. In contrast, deletion of the PxxPxxP motif (Δ47–53) completely abolished this suppression (Fig. 4b). Consistently, overexpressing of ASPΔ47–53 failed to downregulate Ifnb, Isg15, Cxcl5, and Tnf transcription after HSV-1 infection (Fig. 4c). Co-IP assays further demonstrated that the Δ47–53 mutant lost the ability to disrupt the TBK1–IRF3 (Fig. 4d) and STING–TBK1 interactions (Fig. 4e). Together, these results identify the PxxPxxP motif as essential for ASP’s antagonism of host antiviral signaling.

To further define the functional contribution of individual residues within the PxxPxxP motif, we generated point mutants substituting each proline with alanine. Notably, only the Pro47-to-alanine substitution (P47A) abolished ASP’s ability to suppress IFN-β promoter activity (Fig. 4f). In L929 cells, wild-type ASP strongly reduced the phosphorylation of TBK1, IRF3, p38, p65, and ERK, whereas P47A mutant failed to exert this inhibitory effect (Supplementary Fig. 4a). We constructed the HIV-1 P47A pseudovirus, and we initially confirmed that this mutation does not affect the expression of key viral proteins including Env, Pol, and Gag, ensuring that viral entry and assembly functions are fully preserved (Supplementary Fig. 4b). Infection of primary MDMs with HIV-1 P47A or HIV-1Δasp lead to increased phosphorylation of TBK1, IRF3, p38, p65, and ERK compared with the wild-type virus (Fig. 4g) and failed to suppress IFN and cytokine induction (Supplementary Fig. 4c). At the effector level, the P47A mutant failed to repress MX2 transcription without affecting SAMHD1 (Supplementary Fig. 4d, e), a defect in immune evasion that significantly compromised viral replication to levels comparable with HIV-1Δasp throughout the 9-day time course (Supplementary Fig. 4f). Immunofluorescence confirmed enhanced IRF3 nuclear translocation following infection with either mutant (Fig. 4h). Mechanistically, exogenous co-IP in HEK293T cells revealed that wild-type ASP, but not P47A, disrupted TBK1–STING (Fig. 4i) and TBK1–IRF3 interactions (Fig. 4j), and inhibited IRF3 dimerization (Fig. 4k). Endogenous co-IP in HIV-1-infected MDMs confirmed that both ASP deletion and P47A mutation abolished ASP-mediated disruption of the STING–TBK1–IRF3 complex (Supplementary Fig. 4g). Collectively, these results identify Pro47 as a pivotal factor for immunosuppressive activity of the ASP by inhibiting TBK1 signaling.

ASP recruits RNF114 to promote K6-linked ubiquitination of TBK1 at K236
Ubiquitination is a key posttranslational modification regulating innate immune responses. While K63- and K48-linked polyubiquitin chains are generally associated with TBK1 activation and degradation, respectively25, 26, recent studies highlight the regulatory importance of less common linkages, including K6, K11, K27, K29, and K3327. To evaluate the effect of ASP on TBK1 ubiquitination, we coexpressed HA-tagged ubiquitin (wild-type or lysine-specific mutants) with TBK1 in HEK293T cells. Notably, ASP did not alter K63- nor K48-linked ubiquitination but markedly increased K6-linked polyubiquitination of TBK1 (Fig. 5a). This finding was confirmed in L929 cells, where GFP-ASP overexpression strongly promoted K6-linked TBK1 polyubiquitination (Supplementary Fig. 5a). To define the functional relevance, we generated an Ub-K6R mutant plasmid in which lysine 6 was mutated to arginine while all other lysines remained intact. ASP overexpression enhanced both total ubiquitin and K6-linked ubiquitin accumulation on TBK1; however, this effect was completely abolished with the K6R mutant. In contrast, the ASP P47A mutant failed to enhance TBK1 ubiquitination across any condition (Supplementary Fig. 5b).

To identify the E3 ligases involved in ASP-mediated K6-linked ubiquitination of TBK1, we immunoprecipitated Flag-TBK1 from HEK293T cells co-expressing GFP-ASP or GFP control and analyzed the complexes by mass spectrometry. Proteomic profiling revealed significant enrichment of four E3 ligases—EIF3F, RNF114, TUBA1B, and TRIM14—in ASP-expressing cells compared with controls (Fig. 5b). Among these, RNF114 most strongly promoted K6-linked ubiquitination of TBK1 (Fig. 5c and Supplementary Fig. 5c). Critically, this effect was abolished when ubiquitin was mutated to K6R (Fig. 5c). Immunofluorescence combined with co-IP assays further demonstrated that ASP enhances the colocalization and interaction between RNF114 and TBK1, whereas the ASP P47A mutant disrupts this process (Fig. 5d and Supplementary Fig. 5d). Clinically, RNF114 transcript levels were significantly elevated in both ART-treated and treatment-naive cohorts (Fig. 5e), with the latter group exhibiting a positive correlation between RNF114 expression and plasma HIV RNA (Fig. 5f). Moreover, wild-type HIV-1 (but not ASP-deficient virus) strongly induced RNF114 expression (Fig. 5g). Collectively, these findings support data support the model in which ASP facilitates immune suppression by recruiting RNF114 to promote K6-linked ubiquitination of TBK1, thereby enhancing HIV-1 immune evasion.

To assess whether RNF114-mediated ubiquitination disrupts TBK1 function, we overexpressed Myc-tagged RNF114 in HEK293T cells. RNF114 inhibited TBK1-induced IFN-β (Fig. 5h) and ISRE promoter activation (Supplementary Fig. 5e) dose-dependently. Overexpression of RNF114 also significantly reduced the transcription of Ifnb, Isg15, Cxcl5, and Tnf (Fig. 5i and Supplementary Fig. 5f-h) and was accompanied by decreases in phosphorylation of TBK1, IRF3, p38, p65, and ERK (Supplementary Fig. 5i). Co-IP assays revealed that RNF114 disrupted interactions between STING-TBK1, TRAF3-TBK1, and TBK1-IRF3, while suppressing IRF3 dimerization (Supplementary Fig. 5j-m). Conversely, RNF114 depletion restored IFN-I signaling and reversed ASP-mediated suppression (Supplementary Fig. 5n-o). We next evaluated EN219, a small-molecule RNF114 inhibitor, for antiviral efficacy. Primary MDMs were infected with HIV-1 or HIV-1 ASP P47A pseudoviruses and treated with DMSO or EN219 for 1 h. EN219 effectively reversed ASP-mediated suppression of TBK1, IRF3, p38, and ERK phosphorylation (Fig. 5j) and restored IFN-I-related cytokine expression (Supplementary Fig. 6a). Notably, EN219 significantly reduced HIV-1 transcriptional activation in MDMs (Supplementary Fig. 6b), reversed ASP-mediated suppression of IFNG and CD69 mRNA in Jurkat cells (Supplementary Fig. 6c), and inhibited intracellular HIV-1 RNA levels (Supplementary Fig. 6d). Consistent with pseudovirus results, replication-competent HIV-1 infection of primary MDMs under BSL-3 containment showed that EN219 increased transcription of IFNB, ISG15, and TNF (Supplementary Fig. 6e) regardless of viral strain (HIV-1 vs. HIV-1 ASP P47A) and strongly suppressed HIV-1 tat expression (Supplementary Fig. 6f). And EN219 treatment effectively abolished the replication advantage of wild-type, preventing the characteristic surge in viral titers and suppressing replication levels to those comparable to the HIV-1Δasp mutant (Supplementary Fig. 6g). In L929 cells transfected with GFP-ASP or GFP-vector prior to HSV-1 infection, EN219 restored phosphorylation of TBK1, IRF3, p38, p65, and ERK (Supplementary Fig. 6h) and rescued IFN-I–responsive gene expression (Supplementary Fig. 6i). Mechanistically, Co-IP assays demonstrated that EN219 counteracted ASP-induced disruption of STING–TBK1 (Supplementary Fig. 6j) and TBK1–IRF3 (Supplementary Fig. 6k) interactions and restored IRF3 dimerization (Supplementary Fig. 6l).

To identify the TBK1 residue regulated by RNF114, we co-expressed Flag-TBK1 with either Myc-vector or Myc-RNF114 in HEK293T cells. TBK1 immunoprecipitation followed by mass spectrometry revealed that RNF114 overexpression selectively enhanced ubiquitination at Lys236 (K236) (Fig. 5k). Functional validation using a lysine-to-arginine substitution mutant, TBK1 (K236R), revealed that ASP induced K6-linked ubiquitination of wild-type TBK1, but neither ASP nor ASP P47A modified TBK1 (K236R) (Fig. 5l). Similarly, RNF114 promoted total and K6-linked ubiquitination of WT TBK1 but failed to do so for TBK1 (K236R) (Fig. 5m). Functionally, ASP (but not ASP P47A) suppressed IFN-β promoter activity in TBK1-transfected cells, whereas neither ASP nor ASP P47A impaired IFN-β activation driven by TBK1 (K236R) (Fig. 5n). In vitro kinase assays showed that RNF114 suppressed TBK1-mediated phosphorylation of IRF3 but had no effect on phosphorylation by TBK1 (K236R) (Fig. 5o). Likewise, RNF114 inhibited the autophosphorylation of wild-type TBK1 (Supplementary Fig. 6m) and STING phosphorylation (Supplementary Fig. 6n), but these effects were abolished in the K236R context. Moreover, qPCR analyses further confirmed that ASP-mediated suppression of antiviral gene transcription was lost when TBK1 (K236R) was expressed (Fig. 5p and Supplementary Fig. 6o). Collectively, these findings establish that ASP recruits RNF114 to catalyze K6-linked ubiquitination of TBK1 at K236, thereby impairing TBK1 kinase activity and downstream antiviral signaling.

PHD3 promotes the proline hydroxylation of ASP
Proline hydroxylation is a common posttranslational modification that regulates protein activity and signal transduction through oxygen-dependent mechanisms28. Given the critical role of Pro47 in ASP, we hypothesized that this residue serves as a hydroxylation site. Immunoprecipitation assays using an anti-hydroxyproline antibody specifically detected hydroxylated wild-type ASP, but not the P47A mutant (Fig. 6a), indicating site-specific modification. Reciprocal co-IP with anti-GFP beads confirmed Pro47 hydroxylation by Hyp, which was abolished by the P47A mutation (Fig. 6b). Since proline hydroxylation is catalyzed by 2-oxoglutarate- and oxygen-dependent dioxygenases (PHD1, PHD2, and PHD3)29, we coexpressed HA-tagged PHD1, PHD2, or PHD3 with ASP in HEK293T cells. Co-IP revealed a specific interaction between ASP and PHD3, but not with PHD1 or PHD2 (Fig. 6c). Computational protein docking predicted a high-affinity ASP-PHD3 complex (score 0.883), with PLIP analysis identifying multiple hydrogen bonds at the binding interface (Fig. 6d). Validating this interface, mutagenesis of the predicted contact residues in PHD3 attenuated ASP binding in co-ip assays, confirming the structural model (Supplementary Fig. 7a). To evaluate whether HIV-1 infection regulates prolyl hydroxylase expression, RNA-seq of PBMCs from HIV-1-infected individuals showed selective upregulation of PHD3 mRNA (Fig. 6e and Supplementary Fig. 7b). PHD3 expression was consistently upregulated across both ART-treated and treatment-naive patients (Fig. 6f), and crucially, this abundance positively correlated with plasma HIV RNA specifically in the naive group (Fig. 6g). In THP-1 cells, HIV-1 infection selectively enhanced PHD3 expression—an effect abolished in ASP-deficient virus (Fig. 6h)—while PHD1 and PHD2 remained unchanged (Supplementary Fig. 7c). Functionally, PHD3 overexpression in HEK293T cells markedly enhanced hydroxylation of wild-type ASP but not the P47A mutant (Fig. 6i). Subsequent Co-IP revealed that PHD3-mediated hydroxylation promoted both ASP–TBK1 and TBK1–RNF114 interactions, with no effect on the P47A mutant (Fig. 6j, k). Collectively, these findings demonstrate that PHD3-driven hydroxylation of ASP at Pro47 is essential for RNF114 recruitment to TBK1.

To evaluate the functional role of PHD3-mediated hydroxylation in ASP-dependent immune suppression, HIV-1–infected MDMs were treated with the PHD3-specific inhibitor Molidustat. Treatment reactivated multiple signaling pathways, as evidenced by restored phosphorylation of TBK1, IRF3, p38, p65, and ERK (Fig. 6l), and reversed the transcriptional repression of IFNB, ISG15, and TNF (Fig. 6m). Co-IP analysis confirmed that Molidustat disrupted ASP-driven assembly of the TBK1–RNF114 complex (Supplementary Fig. 7d). In a THP-1/Jurkat co-culture model of immune cell interactions, Molidustat abrogated ASP-mediated suppression of IFNG and CD69 expression (Fig. 6n) and significantly reduced viral transcriptional activation (Fig. 6o). These findings were validated in replication-competent HIV-1–infected MDMs under BSL-3 containment (Supplementary Fig. 7e-g). To further define PHD3’s role, we performed siRNA-mediated knockdown in PMs prior to pseudovirus infection. PHD3 silencing restored phosphorylation of TBK1, IRF3, p38, and ERK (Supplementary Fig. 7h), reactivated cytokine expression (Supplementary Fig. 7i), and significantly reduced tat expression (Supplementary Fig. 7j). Collectively, both genetic suppression (siRNA) and pharmacological inhibition (Molidustat) of PHD3 abrogated ASP-mediated immune suppression, establishing PHD3 as a compelling therapeutic target for HIV intervention.

Targeting ASP-mediated immune evasion suppresses HIV-1 replication in vivo
Since ASP suppresses TBK1 activation in vitro, we assessed its role in vivo using humanized peripheral blood lymphocyte (hu-PBL) humanized mouse model generated by adoptive transfer of human PBMCs (Fig. 7a). This model contains predominantly CD4⁺ T cells, along with CD8⁺ T cells and limited monocytes/macrophages, and has been extensively validated for in vivo HIV studies30. Flow cytometry of peripheral blood CD45⁺ cells confirmed successful humanization (Fig. 7b). Mice were intraperitoneally infected with HIV-1. On day 10 post-infection, peripheral blood was collected via the retro-orbital venous plexus before euthanasia and tissue harvesting. ASP deficiency induced robust upregulation of IFNG, IFNB, and ISG15 in the spleen (Fig. 7c), liver (Fig. 7d), and rectal tissues (Fig. 7e), accompanied by elevated serum IFN-γ and IFN-β concentrations (Fig. 7f). This enhanced antiviral response correlated with significantly reduced tissue-associated viral tat mRNA levels (Figs. 7c-e) and serum HIV-1 P24 antigen (Fig. 7f and Supplementary Fig. 8a). To directly assess viral burden in immune tissues, multiplex immunohistochemistry for HIV P24 (viral marker), CD3 (T cells), and CD68 (macrophages) was performed on splenic sections. ASP deficiency markedly reduced P24 expression (Fig. 7g), and colocalization analysis showed substantially decreased P24 association with T cells and macrophages (white arrow), indicating that ASP promotes viral persistence within immune cell niches.

To evaluate potential therapeutic drugs targeting ASP-dependent pathways, hu-PBL mice were infected with HIV-1 and treated daily with either the RNF114 inhibitor EN219 or the PHD3 inhibitor Molidustat (Fig. 7h). By day 10 post-infection, both treatments significantly reduced viral burden in tissues and serum, as evidenced by decreased P24 antigen levels and lower tissue-associated viral loads (Fig. 7i-l and Supplementary Fig. 8b). Importantly, EN219 and Molidustat reproduced the immunostimulatory profile seen in ASP-deficient infections, including increased IFNB and IFNG expression in tissues and elevated serum IFN-β and IFN-γ concentrations (Fig. 7i-l). Multiplex immunohistochemistry further confirmed a pronounced reduction of HIV-1 P24 antigen within tissues from treated mice, demonstrating that inhibition of RNF114 or PHD3 restores antiviral immunity and limits viral persistence (Fig. 7m). These in vivo findings establish that both compounds exert potent antiviral effects during the early stages of HIV-1 infection.

Methods
Ethics statement
Written informed consent for both study participation and the publication of anonymized experimental data was obtained from all adult participants prior to enrollment, following a clear explanation of the study protocol and intended sample use. This research adhered to the ethical guidelines set forth by Fudan University and was reviewed and approved by the Fudan University Ethics Committee (approval number: 2024-Y008-01). Detailed clinical characteristics, including age, sex, CD4+ T cell counts, duration of antiretroviral therapy (ART), and plasma HIV-1 RNA levels, are provided in Supplementary Table 2. The biological sex of all participants was determined via medical records, and participants were recruited randomly based strictly on clinical criteria. All the experiments with mice were conducted in animal care facility of Fudan University and in accordance with the Institutional Animal Care and Use Committee (approval number: 2021-D086-01).

Experimental animal models
6 to 8-week-old male NOD.Cg-PrkdcscidIl2rgcm1cya/Cya (C-NKG) mice were purchased from Shanghai Saiye Biotechnology Co., Ltd. and used to establish the C-NKG-Hu-PBL humanized mouse model. Housing conditions were as follows: temperature 22 ± 2°C, humidity: 50 ± 10%, light and dark cycle: 12 hours light and 12 hours dark, ventilation: approximately 12 cycles per hour of filtered/non-recycled air. For the generation of C-NKG-Hu-PBL mice46, 5 × 106 PBMCs were isolated from healthy human donor blood buffy coats via Ficoll density gradient centrifugation, and subsequently injected intraperitoneally (i.p.) into C-NKG mice in a 200 µL volume using a 1 mL syringe with a 25-gauge needle. Cell engraftment was assessed 14 days post-transplantation. Blood (100 µL) was collected by retroorbital bleeding, and PBMCs were isolated using Ficoll density gradient centrifugation. The cells were then stained with fluorophore-conjugated anti-human CD45 antibodies and analyzed by flow cytometry to confirm successful engraftment. Humanized mice with stable human leukocyte reconstitution were infected intraperitoneally (i.p.) with 5 ng p24 per mouse. Mice infected with 293T mock supernatant served as controls. The entire infection process was carried out in the P3 biosafety laboratory (BSL-3) at Fudan University.

Cells
HEK293T, HeLa, L929, and Vero cells were obtained from the American Type Culture Collection (ATCC). TZM-bl cells were obtained from the NIH HIV Reagent Program. These cell lines, along with primary mouse PMs, were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco). THP-1 and Jurkat cells were obtained from the American Type Culture Collection (ATCC). Primary human PBMCs and MDMs were isolated from healthy donors. These cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium, also supplemented with 10% (v/v) heat-inactivated FBS (Gibco). Both DMEM and 1640 media were supplemented with 100 U/ml penicillin and streptomycin (Hyclone).

Regents and plasmids
The following antibodies were used in this study: anti-TBK1 (3504, 1:2000), anti-phospho-TBK1 (5483, 1:1000), anti-phospho-IRF3 (29047, 1:500), anti-IRF3 (4302, 1:1000), anti-phospho-p65 (3033, 1:1000), anti-p65 (8242, 1:1000), anti-phospho-p38 (9215, 1:1000), anti-p38 (8690, 1:1000), anti-phospho-Erk1/2 (9101, 1:1000), anti-Erk1/2 (4695, 1:1000), all from Cell Signaling Technology; anti-GST (CW0085M, 1:5000) and anti-His (CW0083M, 1:5000), from Abclonal; anti-GFP (50430-2-AP, 1:20000), anti-Myc (16286-1-AP, 1:10000), anti-HA (81290-1-RR, 1:10000), anti-Flag (66008-4-Ig, 1:10000), anti-Lamin B1 (12987-1-AP, 1:1000), anti GAPDH (60004-1-Ig,1:100000), all from Proteintech; anti-p24 (ab32352, 1:1000), anti-CD3 (ab237721, 1:100), anti-CD68 (ab283654, 1:600), from Abcam; anti-gp160 (bs-0786R, 1:1000), anti-SAMHD1 (bsm-61737R, 1:10000), from Bioss; anti-phospho-SAMHD1 (Thr592, 1:500) (TA3508), from Abmart; anti-HIV p51 (40244-T62, 1:1000), anti-RNF1144 (203153-T44, 1:1000), from Sino Biological.

Flag-N-RIG-I, Flag MAVS, Flag-TRAF3, Flag-TBK1, Flag-IRF3, and HA-Ubs were was generously provided by Dr. B. Ge (Tongji University, Shanghai, China). Plasmid expressing Flag-STING was obtained from Dr. B. Sun (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China). Plasmid expressing Myc -EIF3F, Myc-RNF114, Myc-TRIM14 and TUBA1B were constructed and preserved by our lab. Point mutations of the TBK1, as well as the point mutations and truncation mutations of ASP, were performed using a KOD Plus Mutagenesis kit (SMK101, TOYOBO) according to the manufacturer’s protocols. The infectious molecular clone pNL4-3, pYU2 as well as VSV-G-pseudotyped NL4.3-Δenv was obtained from Dr. D. Sauter (Ulm University, Meierhof-strasse, Germany). pNL4-3 (YU2) was generated by replacing the KpnI-to-BamHI fragment within the env gene of the pNL4-3 backbone with the corresponding sequence from the HIV-1 YU2 isolate, as detailed in established protocols47. Site-directed mutagenesis was performed on pNL4-3, pNL4-3(YU2), and NL4.3-Δenv backbones using the Mut Express MultiS Fast Mutagenesis Kit V2 (C215-01, Vazyme) following the protocol provided by the manufacturer. To generate the HIV-1 Δasp mutant, a single nucleotide substitution (TGC to TGA) was introduced at the 12th codon of the asp gene, creating a premature stop codon. Small interfering RNA were synthesized by Tsingke Biotechnology (Beijing, China). The primer sequences used to amplify human and mouse genes were described in Supplementary Table 1.

Virus
HIV-1 and HIV-1Δasp VSV-G-pseudo-typed NL4.3-Δenv viruses were generated in HEK293T cells. Following filtration through a 0.45 μm filter, viral supernatants were concentrated using a virus precipitation reagent (C2901, Beyotime) according to the manufacturer’s protocol. Viral titers were determined in TZM-bl cells using a Luciferase Assay System (E1501, Promega), in accordance with the manufacturer’s instructions. Replication-competent HIV-1, HIV-1Δasp and HIV ASP P47A viruses were produced in HEK293T cells and used for acute infection experiments. Virus titers were determined by p24 capsid protein ELISA, following the manufacturer’s instructions. For HIV-1 pseudo-viruses infection, MDMs, PMs and THP-1 cells were cultured without serum for 12 h and then infected with virus (MOI = 1) for the indicated time. And replication-competent AHIV-1 NL4-3 (YU2) were used for infection at 200 pg of p24 for primary 2 × 106 MDM cells. The experiments involving replication-competent HIV-1 were conducted in the BSL-3 at Fudan University. HSV-1 and VSV-1 were collected from supernatants of infected Vero cells and titrated using Vero cells. HSV-1 and VSV-1 were cultured with L929 (MOI = 10) in DMEM without FBS for 12 h, and then DMEM with 10% FBS for the indicated time.

Isolation of human PBMC, MDMs and mouse PMs
Peripheral blood mononuclear cells (PBMCs) were obtained by isolating mononuclear cells from the blood of healthy volunteers using a human peripheral blood mononuclear cell separation medium (P8610, solarbio), following the manufacturer’s instructions. The isolated PBMCs were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 10 ng/mL human granulocyte-macrophage colony-stimulating factor (hGM-CSF) (300-03, Peprotech) for 3 days. The medium was then replaced to remove non-adherent cells, and the cells were further cultured in RPMI-1640 medium containing 10% FBS and 10 ng/mL hGM-CSF for an additional 3 days. The adherent cells were subsequently detached using 0.25% trypsin and prepared for further assays. For the isolation of mouse peritoneal macrophages (PMs), 4-6-week-old female and male C57BL/6 mice were intraperitoneally (i.p.) injected with 2 mL of 4% Brucella Broth. After 3 days, peritoneal lavage fluid was collected and washed three times with PBS. The peritoneal macrophages were then cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

Quantitative real-time PCR
Cells infected with viruses for the specified time points were lysed using RNAiso Plus (9109, Takara) according to the manufacturer’s protocol. Total RNA (1 µg) was reverse-transcribed into cDNA using the HiScript IV RT SuperMix for qPCR (RT-2GQ, EZB). Quantitative real-time PCR was performed using the Light Cycler (LC480, Roche) and SYBR RT-PCR Kit (Q411-02/03, Vazyme). Gene expression was quantified using the ΔΔCt method, with GAPDH serving as the internal control. Primer sequences for human and mouse genes are listed in Supplementary Table 1.

Immunoprecipitation and western blotting
Cells were cultured and treated for the specified time, then washed three times with cold PBS and lysed in lysis buffer (208256, Beyotime) containing a protease inhibitor cocktail (04693159001; Roche), 1 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM Na3VO4, and 1 mM NaF. After a 30-minute incubation on ice, the lysates were centrifuged at 15,270 x g for 15 minutes at 4°C. The resulting supernatants were incubated with anti-Flag M2 Affinity Gel or Protein A/G Sepharose Fast Flow, along with the appropriate antibodies, at 4°C overnight. The Sepharose-bound samples were then centrifuged and washed three times with ice-cold PBST buffer (1% Triton X-100 in PBS). The precipitates or cell lysates were boiled in 1× SDS loading buffer at 100°C for 10 minutes and analyzed by immunoblotting.

RNA-seq library preparation, sequencing, and data processing
MDMs were infected with HIV-1 or HIV-1Δasp for 24 h, and total RNA was extracted using RNAiso Plus (Takara) following the manufacturer’s instructions. cDNA libraries were constructed and sequenced using an Illumina NovaSeq 6000 platform (Personalbio). Sequencing reads were trimmed to remove poly(A) tails, and low-quality reads were discarded using Cutadapt. High-quality reads were then aligned to the human reference genome (GRCh38) using HISAT2, and gene counts were quantified with HTseq. Gene expression was normalized as fragments per kilobase per million mapped fragments (FPKM) and transformed to log_2 for visualization. Differential gene expression analysis was conducted using DESeq2 with significance thresholds set at fold change > 2 and adjusted P value < 0.05. Volcano plots and heatmaps were generated using ggplot2 and pheatmap R packages, respectively. Gene set enrichment analysis (GSEA) was performed using the GSEA software (Broad Institute). Statistical significance of the GSEA was evaluated using an empirical phenotype-based permutation test, where the nominal P value represents a one-sided test. Enrichment significance was defined by a false discovery rate (FDR) < 0.25 and a nominal P value < 0.05. Adjustments for multiple comparisons in GSEA were applied via the FDR. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, were performed using KEGG Automatic Annotation Server (KAAS). Statistical significance of the KEGG analysis was evaluated using a one-sided hypergeometric test. The resulting nominal P values were adjusted for multiple comparisons using the Benjamini Hochberg FDR method. Enrichment significance for KEGG was defined by an adjusted P value < 0.05.

GST pull down
His or GST proteins were transformed into E. coli BL21(DE3) (Tiangen Biotech), and protein expression was induced by 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; I6758, Sigma-Aldrich) at 30°C for 9 hours. Cells were then sonicated and proteins were purified using Ni-NTA (SA005010, Smart-Lifesciences) or glutathione (SA010010, Smart-Lifesciences) resins. The purified His-fusion proteins or MDM lysates were incubated with GST-fusion proteins conjugated to glutathione beads at 4°C for 4 hours. After centrifugation and washes with cold PBS, the beads were boiled in 1× SDS loading buffer at 100°C for 10 minutes and analyzed by immunoblotting.

Native PAGE and Immunoblotting
Following established principles48, L929, THP-1 and MDM cells (2 × 106) were infected with HSV-1, HIV-1, or HIV-1Δasp pseudo-viruses for the indicated durations. Cells were harvested using ice-cold Native Lysis Buffer (ab156035, Abcam) and centrifuged at 15,270 x g for 10 minutes. The supernatants were quantified and diluted with Native PAGE sample buffer (125 mM Tris–HCl, pH 6.8; 30% glycerol; 0.1% bromophenol blue). A total of 30 µg of protein was loaded onto a pre-run 7.5% native polyacrylamide gel. After electrophoresis, proteins were transferred onto a PVDF membrane and analyzed by immunoblotting.

RNA Interference
siRNAs were designed and synthesized by the Tsingke (China); siRNA sequences are listed in the Supplementary Table 1. siRNAs against TBK1 and RNF114 and negative control were obtained from Santa Cruz. PMs were transfected with siRNA using RNAi MAX reagent (Invitrogen) according to manufacturer’s instructions.

Dual-luciferase reporter assay
HEK293T cells were transiently transfected with pRL-IFN-β–Luc or pRL-ISRE–Luc, pRL-TK, and the indicated plasmids for 24 h. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (RG028, Beyotime) according to the manufacturer’s instructions.

In vitro TBK1 Kinase Assay
The kinase assays were adapted from established method49, with minor modifications. L929 cells transfected with Flag-TABK1 and other plasmids for 36 h were lysed with cell lysis buffer. Lysate was incubated with anti-flag magnetic beads (Millipore) for 2 h at 4℃. Beads were washed twice with cell lysis buffer and then twice with kinase buffer (CST). Kinase reactions were performed by incubation of washed beads with kinase buffer, 1 mM ATP (CST) and 1mg recombinant IRF3, TBK1 and STING as the substrate at 30℃ for 2 h. Samples were separated on SDS-PAGE, and analyzed by immunoblot.

Immunofluorescence and Confocal Microscopy
Cells were seeded onto confocal dishes and infected with viruses for the specified time points, fixed with 4% formaldehyde for 15 minutes, and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Blocking was performed with 5% bovine serum albumin (BSA) for 1 hour. The samples were then incubated with primary antibodies overnight at 4°C, followed by three washes with PBS containing 0.1% Tween-20. Secondary antibodies were applied for 1 hour at 37°C, and the nuclei were counterstained with DAPI for 5 minutes. Fluorescence images were acquired using a Leica SP8-STED confocal microscope.

Protein–Protein Docking
Protein interaction structures were predicted using AlphaFold3, followed by molecular docking analysis conducted according to established protocols50, 51, 52. Docking was performed with HDOCKlite v1.1 in global mode, and the top-ranked complex based on docking and confidence scores was selected for further analysis. Binding free energy was calculated using the MM/GBSA module of HawkDock. Protein–protein interaction interfaces were characterized with PLIP and visualized using PyMOL.

Multiplex Immunofluorescence Histochemistry (mIHC)
Spleen tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm thickness. After deparaffinization and rehydration, antigen retrieval was performed in citrate buffer (pH 6.0) at 95 °C for 20 min. Sections were blocked with 5% BSA and incubated with primary antibodies against human CD3, CD68, and HIV-1 P24 overnight at 4 °C, followed by incubation with appropriate fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI. After mounting, images were captured using a confocal fluorescence microscope (Leica TCS SP8). Quantification of fluorescence intensity was performed using ImageJ software (NIH).

Statistical analysis
Data are presented as mean ± SD. GraphPad Prism 8 software was used for statistical analysis and data presentation. For comparisons between two independent groups, two-tailed unpaired Student’s t-test was used. For comparisons among three or more groups with a single independent variable, a one-way ANOVA was performed, followed by Dunnett’s or Tukey’s post hoc test for multiple comparisons. For experiments involving two independent variables, a two-way ANOVA was utilized, followed by Sidak’s or Dunnett’s multiple comparisons test. Correlation analysis was performed using Linear regression. Statistical significance is indicated in the figure legends: *P < 0.05, **P < 0.01, ***P < 0.001. Number of animals and replicates for each experiment are shown in figures or indicated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Lin et al. describes a novel mechanism by which HIV-1 evades host innate immunity through its\nantisense protein (ASP). The authors demonstrate that ASP undergoes PHD3-mediated hydroxylation at Pro47, which\nenables recruitment of the E3 ligase RNF114 to promote K6-linked ubiquitination of TBK1, thereby suppressing interferon\nresponses. The work includes comprehensive mechanistic studies, clinical correlations, and in vivo validation using\nhumanized mouse models. While the findings are potentially significant for understanding HIV-1 persistence and identifying\nnew therapeutic targets, several critical issues require attention before publication.\nMAJOR COMMENTS\n1) While the authors claim that viral entry remains unchanged between wild-type HIV-1 and HIV-1Δasp based on\npseudovirus experiments (Extended Data Fig. 2a), the construction and characterization of HIV-1Δasp virus requires more\ndetailed description. The method used to disrupt the asp gene is unclear (complete deletion? frameshift mutation? stop\ncodon insertion?). Given that ASP overlaps with env in antisense orientation, how did the authors ensure that asp disruption\ndid not affect other viral proteins? The authors should compare viral protein expression profiles (Gag, Pol, Env, accessory\nproteins) in both transfected cells and culture supernatants among wild-type, asp-deleted, and asp-mutant (P47A) pNL4-3\nconstructs.\n2) While Figures 1a, 5e, and 6f provide valuable clinical data, the ambiguous definitions of \"Latency\" and \"AIDS\" may\nconfuse readers. In latent infection, viral genome transcription should be suppressed by histone methylation - are the authors\nsuggesting that asp transcription bypasses this epigenetic silencing? The authors must provide comprehensive clinical\nparameters for each patient group, including viral reservoir size, viral load, CD4+ T cell counts, duration of ART, and specific\ncriteria for patient classification.\n3) The extensive use of GFP-tagged ASP raises concerns about protein functionality. Fusion with such a large tag (27 kDa)\nmay alter ASP's native subcellular localization and protein interactions. The authors should generate ASP constructs with\nsmaller tags (HA or FLAG) and compare their localization and functional properties with GFP-ASP to validate their\nexperimental system.\nMINOR COMMENTS\n-Figures 2e and 6d show computational docking models. If these are to be presented in main figures, experimental\nvalidation through mutagenesis of predicted interface residues followed by co-immunoprecipitation is essential. Also,\nremove unused legend items (e.g., \"Water\").\nMultiple Western blots lack GFP bands for empty vector controls (e.g., Figures 1d, 2b, 2c, 2g, 3d-h, 3l, 4d, 4e, 4i-k, 5a, 5l, 6a,\n6b, 6i, 6k).\n-Co-immunoprecipitation experiments must include appropriate input controls showing the target proteins. Without these,\ninteraction changes cannot be properly assessed (e.g., missing HA blots in Figures 3d-g, 4d-e, 4i-k; missing GFP blot in\nFigure 6j).\n-Band quantification is required when claiming reduced protein levels or phosphorylation (e.g., p-IRF3 in Figure 3c).\n-Typographical errors requiring correction:\nFigure 2i and 4a are inconsistent (should be 1-62, 1-84, not 1-64, 1-82).\nFigure 2j left/right panels show different numbering (297-713 vs 382-713).", "reviewer_2": "In this study, Xinguang Lin and colleagues, uncover a novel mechanism by which HIV-1 evades the host immune response\nthrough the activity of its antisense protein ASP. The authors show that ASP inhibits IFN-I signaling by targeting TBK1.\nSpecifically, ASP is hydroxylated at Pro47 by the host enzyme PHD3, which enables recruitment of the E3 ubiquitin ligase\nRNF114 to TBK1. RNF114 then mediates K6-linked ubiquitination of TBK1 at Lys236, inhibiting its activation and\ndownstream IFN-I production. Mutation of ASP at Pro47 or inhibition of PHD3 or RNF114 through specific drugs, improves\nantiviral immunity and reduces viral replication in humanized mice, underscoring the functional importance of this pathway.\nASP has been implicated in processes such as autophagy induction, transcriptional regulation, and latency maintenance,\nand its expression elicits both cellular and humoral immune responses in infected individuals. However, its precise function\nin HIV-1 pathogenesis remains incompletely understood. This is an interesting study that provides new mechanistic insight\ninto ASP) function. The biochemical work is detailed and overall convincing.\nHowever, several aspects of the functional and virological analyses require further clarification and experimental support.\nThe effects reported in the infection and replication assays are modest, and more rigorous, quantitative analyses are needed\nto substantiate the proposed antiviral and immunosuppressive roles of ASP. In addition, the interpretation of latency in ART-\ntreated individuals, patient characterization, viral tropism, and potential interactions of ASP with known HIV restriction factors\nshould be refined.\n• Replication assay: Referring to infection with a single-round virus as “replication” is incorrect. A multi-day time-course with\na fully replication-competent virus is needed to validate replication effects of ASP deletion, mutation, or inhibitor treatment.\n• Replication readout: Measuring Tat transcript levels by qPCR is not a reliable indicator of replication. p24 expression\nshould be quantified by ELISA or flow cytometry at several time points.\n• Latency interpretation: ART-treated individuals should not be referred to as having “latent infection,” as their viremia is\npharmacologically suppressed. Latency should be studied using appropriate models such as GKO or pMorpheus dual\nFluorescent viruses, that allow the discrimination between productive and latent infection\n• Patients’ characterization: Providing additional clinical and immunological information would greatly enhance the\ninterpretability and reproducibility of the results. Specifically, the authors are encouraged to clarify how the AIDS stage was\ndefined and to include data on CD4⁺ T cell counts, CD4/CD8 ratios, plasma viral load (viremia), and any opportunistic\ninfections. Moreover, details regarding antiretroviral therapy (ART)—including treatment duration, current immuno-virological\nstatus, and the specific ART regimens or drug classes used—would be highly informative.\n• Virus tropism: NL4.3 is an X4-tropic virus and cannot efficiently infect macrophages, which require an R5-tropic virus. This\nissue needs to be clarified or experimentally corrected.\n• HIV restriction factors: are specific HIV restriction factors that are ISGs (MX2 for example) upregulated in the absence of\nASP? Is phosphorylation of SAMHD1 decreased in the presence of ASP?\nMinor Comments:\nFigure 1\n• Figure 1A: The label “Latency” is not accurate. These samples represent patients on ART with pharmacologically\nsuppressed viremia, which is not equivalent to latency. The observed ASP upregulation therefore does not necessarily\nindicate a role in viral persistence.\n• Figure 1D: The expression “strongly inhibited” seems overstated. Please either moderate the language or provide\nquantitative data showing the actual fold reduction in phosphorylation induced by ASP.\n• Figure 1G: The immunofluorescence results are presented qualitatively. Quantification of fluorescence intensity or the\nproportion of positive cells would strengthen the conclusions.\n• Figure 1I: The Western blot for pERK should be replaced with a higher-quality image to allow clearer interpretation.\n• Figure 1L: The Western blot suggests that IRF3 nuclear translocation is enhanced mainly at 4 h. Please include\nquantification to support this observation.\nFigure 2\n• Figure 2F: The immunofluorescence data should also be quantified rather than presented solely as representative images.\n• Figure 2G: The Co-IP Western blot appears unconvincing in its current form. A clearer, better-resolved blot or additional\nreplicates would strengthen the conclusion.\nFigure 3\n• Figure 3N: Please provide Western-Blot to show TBK1 knock-down\nFigure 6\n• A higher-quality version of the anti-GFP Western blot would improve clarity and the reliability of interpretation.\nFigure 7\n• HIV infection in mice should be followed at different time point showing plasma viral load\nLine 26: why the authors state “elevated ASP and PHD3 transcript levels are negatively correlated with immune response in\nART-treated patients during latency”? please clarify."}, "author_response": "Dear Reviewer, Thank you for your continued review of our work. We have carefully considered all\nyour comments and suggestions, which have been invaluable to us. In this revision,\nwe have addressed each of your questions in detail. We believe the updated\nmanuscript fully resolves the concerns raised. All modifications have been highlighted\nin red for easy reference.\nReviewers Comments:\nReviewer #1 (Remarks to the Author):\nThis manuscript by Lin et al. describes a novel mechanism by which HIV-1 evades\nhost innate immunity through its antisense protein (ASP). The authors demonstrate\nthat ASP undergoes PHD3-mediated hydroxylation at Pro47, which enables\nrecruitment of the E3 ligase RNF114 to promote K6-linked ubiquitination of TBK1,\nthereby suppressing interferon responses. The work includes comprehensive\nmechanistic studies, clinical correlations, and in vivo validation using humanized\nmouse models. While the findings are potentially significant for understanding HIV-1\npersistence and identifying new therapeutic targets, several critical issues require\nattention before publication.\nWe thank the reviewer 1 for the thorough assessment and constructive feedback, and\nwe have addressed all the issues raised in the revised manuscript.\nMAJOR COMMENTS:\n1. While the authors claim that viral entry remains unchanged between wild-type\nHIV-1 and HIV-1Δasp based on pseudovirus experiments (Extended Data Fig. 2a),\nthe construction and characterization of HIV-1Δasp virus requires more detailed\ndescription. The method used to disrupt the asp gene is unclear (complete deletion?\nframeshift mutation? stop codon insertion?). Given that ASP overlaps with env in\nantisense orientation, how did the authors ensure that asp disruption did not affect\nother viral proteins? The authors should compare viral protein expression profiles\n(Gag, Pol, Env, accessory proteins) in both transfected cells and culture supernatants\namong wild-type, asp-deleted, and asp-mutant (P47A) pNL4-3 constructs.\nWe thank the reviewer for this critical comment. We fully recognize that the asp gene\noverlaps with the env gene in the antisense orientation, and ensuring the integrity of\nthe Envelope glycoprotein is paramount for interpreting our results.1.\nTo address the concern of the reviewer regarding the construction method, we have\nexpanded the Methods section to provide specific details. Specifically, we introduced\na single nucleotide substitution within the 12th codon of the asp gene in the VSV-G-\npseudotyped NL4.3-Δenv as well as pNL4-3 backbone. This mutation converts a\nCysteine codon (TGC) into a premature Stop codon (TGA), resulting in the early\ntermination of ASP translation. Crucially, due to the specific reading frame overlap,\nthis nucleotide change results in a synonymous (silent) mutation in the env gene on\nthe opposite strand. Therefore, while ASP expression is abolished, the amino acid\nsequence of the Envelope glycoprotein remains 100% identical to the wild-type.\nAccordingly, we have updated the Materials and Methods section to include the\ndetailed cloning strategy. As requested, we have empirically verified this design by\ncomparing viral protein profiles. As shown in Extended Data Fig. 4b, the expression\nlevels of Env (gp160), Pol (p51), as well as Gag (p24), are indistinguishable between\nthe wild-type, asp-deleted, and asp-mutant (P47A) pNL4-3 constructs, confirming\nthat the mutation caused no off-target effects on viral entry or assembly.\nResponse Figure 1. HIV-1Δasp and HIV-1\nP47A do not alter the expression of viral\nproteins.\na, Immunoblot analysis of cell lysates and\nsupernatants from 293T cells transfected with\nWT, HIV-1Δasp, or HIV-1 P47A constructs,\ndetected using antibodies against Env (gp160), Pol (p51), and Gag (p24).\n2. While Figures 1a, 5e, and 6f provide valuable clinical data, the ambiguous\ndefinitions of \"Latency\" and \"AIDS\" may confuse readers. In latent infection, viral\ngenome transcription should be suppressed by histone methylation - are the authors\nsuggesting that asp transcription bypasses this epigenetic silencing? The authors must\nprovide comprehensive clinical parameters for each patient group, including viral\nreservoir size, viral load, CD4+ T cell counts, duration of ART, and specific criteria\nfor patient classification.\nWe sincerely thank the reviewer for this constructive critique regarding the clinical\ndata and the mechanistic interpretation of asp transcription. We have addressed these\npoints by significantly expanding our clinical cohort and refining our mechanistic\nmodel.\nTo further strengthen the robustness of our conclusions and mitigate the potential\nheterogeneity inherent in clinical data, we proactively collected additional clinical\nsamples, ensuring the reliability and solidity of our findings. The cohort now includes\nensure precision, we adjusted our qPCR methodology to measure the absolute copy\nnumber of asp transcripts per µg of cDNA (rather than relative quantification). As\nshown in Fig. 1a, we observed substantial levels of asp transcription in both groups.\nStrikingly, even in ART-suppressed patients where Gag expression is minimal, asp\ntranscripts remain detectable at significant levels. Thus, we confirm that asp\nmaintains active transcription and exerts important biological functions in patients,\nirrespective of whether they are undergoing ART or remain treatment-naive.\nTo avoid ambiguity, we have categorized patients strictly based on clinical treatment\nstatus and CDC criteria, defining Group 1 (ART-Suppressed) as individuals on long-\nterm ART with undetectable plasma viral load and Group 2 (Treatment-Naive) as\nviremic individuals with active viral replication, and detailed clinical parameters\nincluding CD4 count, ART duration, and plasma HIV RNA are provided in Extended\nData Table 2.\nRegarding the potential bypass of epigenetic silencing, we propose a context-\ndependent model that aligns with both the literature and our experimental data. The\ndetection of asp in individuals on suppressive ART is consistent with the distinct\nbiology of the HIV-1 antisense lineage. Unlike the sense strand (Gag/Pol) driven by\nthe 5' LTR, asp is transcribed from the antisense promoter in the 3' LTR. Thus, it is\nnot necessarily subject to the same epigenetic silencing (histone methylation) that\nsuppresses the 5' LTR. Most notably, cumulative studies have provided definitive\ntranscriptional evidence of ASP in patients. Mancarella et al. first confirmed the\nexistence of stable, cell-associated transcripts specifically containing the ASP open\nreading frame (ORF) in HIV-1 infected individuals. Building on this foundation1,\nCapoferri et al. recently successfully sequenced these antisense transcripts directly\nfrom the PBMCs of ART-suppressed individuals, confirming their persistence in vivo2.\nFurthermore, Waast et al. utilized strand-specific ddPCR to demonstrate that antisense\nRNAs are not only present but can exist at high copy numbers in patient splenocytes,\nsometimes exceeding sense transcripts3. These findings unequivocally validate the\nactive transcription of the asp gene in our clinical cohorts.\nHowever, we also observed appreciable asp transcription in treatment-naive patients.\nThis high-level expression during active viral replication suggests that ASP serves a\nfunction beyond merely silencing the virus. Based on the data presented in this\nmanuscript, we propose that the high levels of asp in these patients serve to actively\nsuppress host innate immunity. By hijacking the TBK1 pathway and inhibiting the\ndownstream IFN response, ASP facilitates an environment of immune suppression,\nallowing the virus to evade host surveillance and propagate efficiently.\nTherefore, ASP plays a dual role: it contributes to the regulation of viral transcription\nin the reservoir (as supported by literature) while simultaneously acting as a potent\ninnate immune antagonist to facilitate immune evasion, a mechanism we elucidate in\nthis study.\nResponse Table 1: Demographic and clinical characteristics of HIV-1 infected study\nparticipants\nCD4+ Plasma Duration\nAge CD4/CD8\nPatient ID Sex Count HIV-1 RNA of ART\n(yrs) Ratio\n(cells/µL) (copies/mL) (Years)\nART-\nSuppressed\nDonor 1 49 Male 147 0.22 < 20 1.6\nDonor 2 30 Male 124 0.29 < 20 2.7\nDonor 3 71 Male 255 0.16 < 20 7.2\nDonor 4 39 Male 706 0.89 < 20 6.3\nDonor 5 35 Male 320 0.39 < 20 4.6\nDonor 6 68 Female 497 0.97 < 20 1.5\nDonor 7 71 Male 465 1.04 < 20 25.7\nDonor 8 46 Male 207 0.28 < 20 13.6\nDonor 9 55 Male 1140 1.63 < 20 9.8\nDonor 10 62 Male 581 0.77 < 20 4.3\nDonor 11 43 Male 816 0.48 < 20 5\nDonor 12 75 Male 457 0.64 < 20 3.8\nDonor 13 26 Male 375 0.4 < 20 1.4\nDonor 14 78 Female 372 1.35 54^a 11.3\nDonor 15 62 Male 489 0.36 < 20 3.6\nDonor 16 43 Male 590 1.66 < 20 1.9\nDonor 17 67 Male 827 1.15 42^a 0.1\nTreatment-\nNaive\n>\nDonor 1 56 Female 13 0.07 N/A\n10,000,000\nDonor 2 45 Male 34 0.18 89,000 N/A\nDonor 3 55 Female 33 0.05 339,000 N/A\nDonor 4 29 Male 12 0.03 654,000 N/A\nDonor 5 49 Female 5 0.04 1,220,000 N/A\nDonor 6 35 Male 14 0.13 195,000 N/A\nDonor 7 66 Male 566 0.96 1,240 N/A\nDonor 8 36 Male 108 0.09 414,000 N/A\nDonor 9 32 Male 587 0.71 13,500 N/A\nDonor 10 26 Male 571 1.08 1,140 N/A\n3. The extensive use of GFP-tagged ASP raises concerns about protein functionality.\nFusion with such a large tag (27 kDa) may alter ASP's native subcellular localization\nand protein interactions. The authors should generate ASP constructs with smaller tags\n(HA or FLAG) and compare their localization and functional properties with GFP-\nASP to validate their experimental system.\nWe thank the reviewer for this critical suggestion. We fully appreciate that the\nrelatively large size of the GFP moiety (27 kDa) could potentially introduce steric\nhindrance or artifactual localization, especially for a small viral protein like ASP. To\nrigorously validate our system, we constructed HA- and Flag-tagged ASP and\ncompared their subcellular localization and inhibitory functions with the original\nGFP-ASP construct.\nFirst, we confirmed the expression of HA-ASP and Flag-ASP by Western Blotting\n(Extended Data Fig. 3a). Next, using confocal microscopy, we examined their spatial\ndistribution. As shown in Extended Data Fig. 3b, both HA- and Flag-tagged ASP\nspecifically co-localized with TBK1 in the cytoplasm, appearing as distinct yellow\npuncta in the merged images. This pattern was indistinguishable from that observed\nwith GFP-ASP.\nTo verify functional preservation, we repeated the reporter assays. Both HA- and\nFlag-tagged ASP variants significantly inhibited TBK1-mediated IFN-β promoter\nactivation and suppressed ISRE-luciferase activity, demonstrating efficacy\ncomparable to the GFP-ASP construct (Extended Data Fig. 3c, d).\nCollectively, these data demonstrate that the localization pattern and the\nimmunosuppressive function of ASP are independent of the tag. The use of smaller\ntags (HA/Flag) fully recapitulates the phenotype observed with GFP-ASP, validating\nthe physiological relevance of our experimental system.\nResponse Figure 2. The GFP tag does not alter the intracellular localization or immunosuppressive\nfunction of ASP.\na, Immunoblot analysis of 293T cells transfected with GFP-ASP, HA-ASP, or Flag-ASP constructs,\ndetected using their respective tag-specific antibodies. b, Confocal microscopy comparing the\nsubcellular localization of GFP-ASP, HA-ASP, and Flag-ASP in HeLa cells, co-stained for\nendogenous TBK1 (scale bar, 20 μm). c–d, Luciferase reporter assays comparing the inhibitory\neffects of GFP-ASP, HA-ASP, and Flag-ASP on TBK1-induced IFNB (c) and ISRE (d) promoter\nactivity in HEK293T cells (n = 3 independent biological replicates). Data are means ± SD. *P <\n0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s t-test).\nMINOR COMMENTS:\n1. Figures 2e and 6d show computational docking models. If these are to be presented\nin main figures, experimental validation through mutagenesis of predicted interface\nresidues followed by co-immunoprecipitation is essential. Also, remove unused\nlegend items (e.g., \"Water\").\nWe thank the reviewer for this constructive suggestion to experimentally validate our\ncomputational predictions. We have now performed extensive mutagenesis studies to\nrigorously corroborate the docking models presented in both Fig. 2e and Fig. 6d.\nFirst, regarding the ASP-TBK1 interface (Fig. 2e), our structural analysis highlighted\na critical interaction network driven by key TBK1 residues, including Glu561 (salt\nbridge), as well as Asp586, Gln565, Glu579, Asn578, and Arg574 (hydrogen bonds).\nConsistent with these predictions, Co-IP results demonstrated that specific point\nmutations at these sites markedly attenuated the interaction with ASP compared to the\nwild-type kinase (Revised Fig. 2e).\nSimilarly, for the ASP-PHD3 interaction (Fig. 6d), we targeted the critical PHD3\nresidues identified by our docking model as forming hydrogen bonds and salt bridges.\nCo-IP assays confirmed that mutations at these predicted interface sites weakened the\nbinding affinity between PHD3 and ASP to varying degrees (Extended Data Fig. 7a).\nThese combined experimental data provide compelling physical evidence supporting\nthe accuracy of the molecular interfaces proposed in our computational models.\nRegarding the figure presentation, the unused \"Water\" label has been removed as\nrequested. Furthermore, to enhance visual clarity and focus on biologically relevant\nfeatures, we have streamlined the annotations in both figures to exclusively display\nthe critical interactions (hydrogen bonds and salt bridges) that were experimentally\nvalidated. All non-essential labels have been excluded.\nResponse Figure 3. Mutational analysis validates the predicted interaction interfaces of ASP with\nTBK1 and PHD3.\na, Co-IP analysis of 293T cell lysates co-transfected with ASP and Flag-tagged WT or mutant\nTBK1 constructs (Glu561, Asp586, Gln565, Glu579, Asn578, Arg574). b, Co-IP analysis of 293T\ncell lysates co-transfected with ASP and HA-tagged WT or mutant PHD3 constructs targeting\npredicted interface residues (n = 3 independent times with similar results).\n2. Multiple Western blots lack GFP bands for empty vector controls (e.g., Figures 1d,\n2b, 2c, 2g, 3d-h, 3l, 4d, 4e, 4i-k, 5a, 5l, 6a, 6b, 6i, 6k).\nWe appreciate the reviewer’s emphasis on data completeness. We acknowledge the\nomission of GFP bands for Empty Vector (EV) controls in the initial submission,\nwhich was originally intended to optimize figure layout given the significant\nmolecular weight difference between the fusion protein and free GFP (~27 kDa).\nTo strictly adhere to the reviewer’s request and ensure the highest standard of data\npresentation, we have performed independent repeat experiments for the majority of\nthe indicated panels (Figs. 2b, 2c, 2g, 3d–h, 4d, 4e, 4i–k, 6a, 6b, 6i). These revised\nfigures now explicitly display the GFP bands for the EV controls.\nRegarding Fig. 5a, we have retained the cropped presentation in the main figure to\nmaintain visual clarity and focus; however, we emphasize that the full, uncropped\nscans for this blot—and indeed for all Western blots presented in this manuscript—are\ncomprehensively provided in the Source Data file. This allows for independent\nverification of specificity and expression levels across all experimental conditions.\n3. Co-immunoprecipitation experiments must include appropriate input controls\nshowing the target proteins. Without these, interaction changes cannot be properly\nassessed (e.g., missing HA blots in Figures 3d-g, 4d-e, 4i-k; missing GFP blot in\nFigure 6j).\nWe sincerely thank the reviewer for this crucial comment. We fully recognize that\ninput controls are indispensable for the accurate assessment of protein interactions.\nBeyond addressing the specific panels noted (Fig. 3d-g, 4d-e, 4i-k, and 6j), we\nconducted a comprehensive audit of all main and supplementary figures. We\nidentified and rectified similar omissions in other panels (Extended Data Fig. 5j-m) to\nensure consistency throughout the manuscript. Consequently, all Co-IP datasets\npresented in the revised submission now include the corresponding input controls,\nensuring full data completeness and rigor.\nResponse Figure 4. 3d–f, Co-IP assays in HEK293T cells evaluating the interaction of Flag-\nTBK1 with IRF3 (3d), STING (3e), and TRAF3 (3f) following overexpression of GFP-ASP. 3g,\nCo-IP analysis of IRF3 dimerization in HEK293T cells co-expressing GFP-ASP. 3h, Co-IP assays\nin L929 cells overexpressing ASP and stimulated with HIV-1, to assess endogenous STING–\nTBK1–IRF3 complex formation. 3k, Co-IP assays in MDMs infected with wild-type HIV-1 or\nHIV-1Δasp to assess endogenous TRAF3–TBK1–IRF3 complex formation. 4d-e, Co-IP assays in\nHEK293T cells to determine the effect of PxxP motif deletion in ASP (ASPΔ47-53) on the\ninteractions between TBK1 and STING (4d) and TBK1 and IRF3 (4e). 4i–k, Co-IP assays in\nHEK293T cells examining the effect of wild-type ASP or ASP P47A on TBK1 binding to STING\n(4i), TBK1 binding to IRF3 (4j), and IRF3 dimerization (4k). S5j-m, Co-IP analysis in HEK293T\ncells overexpressing Myc-RNF114 to assess the interactions between TBK1 and STING (S5j),\nTRAF3 (S5k), IRF3 (S5l), as well as IRF3 dimerization (S5m).\n4. Band quantification is required when claiming reduced protein levels or\nphosphorylation (e.g., p-IRF3 in Figure 3c).\nWe appreciate the suggestion regarding data quantification. Accordingly, we have\nperformed densitometric analysis for all phosphorylation bands presented in the main\nfigures using ImageJ. Specifically, phosphorylated protein levels were normalized to\nthe corresponding total protein levels to determine relative activation. To allow for\ndirect visualization of these changes, the normalized numerical values have been\nexplicitly annotated adjacent to the respective blots. Furthermore, we extended this\nquantitative approach to the nuclear translocation assays, subjecting the fractionation\ndata to densitometric analysis to further substantiate the results.\n5. Typographical errors requiring correction:\nFigure 2i and 4a are inconsistent (should be 1-62, 1-84, not 1-64, 1-82).\nFigure 2j left/right panels show different numbering (297-713 vs 382-713).\nWe apologize for these labelling inconsistencies and thank the reviewer for their\nmeticulous attention to detail. We have corrected Figs 2i and 4a to accurately reflect\nthe residue ranges (1–62 and 1–84). Similarly, the numbering in Fig 2j has been\nunified to consistently denote the correct domain boundaries across all panels. These\nerrors have been rectified in the revised figures.\nWe thank the reviewer for their time and constructive comments. We hope these\nrevisions meet your expectations.\nReviewer #2 (Remarks to the Author):\nIn this study, Xinguang Lin and colleagues, uncover a novel mechanism by which\nHIV-1 evades the host immune response through the activity of its antisense protein\nASP. The authors show that ASP inhibits IFN-I signaling by targeting TBK1.\nSpecifically, ASP is hydroxylated at Pro47 by the host enzyme PHD3, which enables\nrecruitment of the E3 ubiquitin ligase RNF114 to TBK1. RNF114 then mediates K6-\nlinked ubiquitination of TBK1 at Lys236, inhibiting its activation and downstream\nIFN-I production. Mutation of ASP at Pro47 or inhibition of PHD3 or RNF114\nthrough specific drugs, improves antiviral immunity and reduces viral replication in\nhumanized mice, underscoring the functional importance of this pathway. ASP has\nbeen implicated in processes such as autophagy induction, transcriptional regulation,\nand latency maintenance, and its expression elicits both cellular and humoral immune\nresponses in infected individuals. However, its precise function in HIV-1 pathogenesis\nremains incompletely understood. This is an interesting study that provides new\nmechanistic insight into ASP) function. The biochemical work is detailed and overall\nconvincing.\nHowever, several aspects of the functional and virological analyses require further\nclarification and experimental support. The effects reported in the infection and\nreplication assays are modest, and more rigorous, quantitative analyses are needed to\nsubstantiate the proposed antiviral and immunosuppressive roles of ASP. In addition,\nthe interpretation of latency in ART-treated individuals, patient characterization, viral\ntropism, and potential interactions of ASP with known HIV restriction factors should\nbe refined.\nWe sincerely thank Reviewer 2 for the constructive comments, and we have fully\naddressed the concerns regarding viral replication assays, patient characterization,\nlatency, and viral tropism by conducting rigorous additional experiments and\nproviding detailed clarifications to substantiate the function of ASP.\n1. Replication assay: Referring to infection with a single-round virus as “replication”\nis incorrect. A multi-day time-course with a fully replication-competent virus is\nneeded to validate replication effects of ASP deletion, mutation, or inhibitor treatment.\nWe thank the reviewer for this critical correction regarding terminology. We agree that\nour initial single-round assays specifically reflected viral transcriptional activation\nrather than spreading infection, and we have clarified this distinction in the revised\nmanuscript.\nTo address the point regarding physiological validation, we performed multi-day\ntime-course experiments (Days 0, 3, 6, and 9) using fully replication-competent HIV-\n1 (NL4-3 YU2), an experimental timeline adopted from recent research characterizing\nsustained infection kinetics in MDMs4. We systematically investigated the impact of\nASP deletion, specific mutation, and pharmacological inhibition on HIV-1 replication\nkinetics. These new results have been incorporated into the revised manuscript (Fig.\n1m, Extended Data Fig. 4f, 6g, 7g).\nWe first established the baseline requirement for ASP in viral spreading. While the\nwild-type replicated efficiently, exhibiting a rapid surge in viral titers that peaked at\nDay 6, the ASP-deficient mutant (HIV-1Δasp) showed significantly attenuated\nreplication kinetics (Fig. 1m). Throughout the 9-day course, the viral yield of the HIV-\n1Δasp remained markedly lower than that of the wild-type virus, confirming that ASP\nis essential for optimal viral propagation in a physiological context.\nTo determine if this pro-viral function relies on the specific interaction motif\nidentified in our structural analysis, we assessed the replication capacity of the P47A\npoint mutant. As shown in Extended Data Fig. 4f, the P47A mutant failed to replicate\nefficiently, effectively phenocopying the growth defect observed in the HIV-1Δasp.\nThis provides definitive genetic evidence that the P47 residue is indispensable for\nASP-mediated viral replication.\nWe next investigated the functional requirement of the RNF114 signaling axis and\ntreated HIV-1 infected cells with EN219. In the vehicle control group, WT HIV-1\nshowed robust replication. However, treatment with EN219 effectively abrogated this\nreplication advantage, preventing the characteristic surge in viral titers and\nsuppressing replication levels to those comparable to the HIV-1Δasp (Extended Data\nFig. 6g). This confirms that ASP relies on RNF114 activity to facilitate viral spread.\nFinally, to verify the involvement of the downstream effector PHD3, we treated the\ninfection system with the specific inhibitor Molidustat. Consistent with the RNF114\nblockade results, Molidustat treatment significantly suppressed the efficient\npropagation of HIV-1 (Extended Data Fig. 7g). The inhibitor reversed the elevation in\nviral titers typically observed at peak infection (Day 6), demonstrating that the\nenzymatic activity of PHD3 is critical for ASP-driven replication.\nCollectively, these comprehensive replication data—spanning genetic deletion, point\nmutation, and pharmacological intervention—provide robust, orthogonal evidence\nthat ASP promotes HIV-1 replication through a P47-dependent mechanism involving\nthe RNF114 and PHD3 signaling pathways.\nResponse Figure 5. ASP\npromotes HIV-1 replication via a\nP47-RNF114-PHD3 axis.\na, Replication kinetics of wild-\ntype (WT) and asp-deficient\n(HIV-1Δasp) viruses (pNL4-3\nYU2) in primary human MDMs,\nquantified by measuring p24\nlevels in supernatants at Days 0, 3,\n6, and 9. b, Replication kinetics\nanalysis comparing WT, HIV-\n1Δasp, and HIV-1 P47A, monitoring viral production over a 9-day time course to assess the\nimpact of the P47 residue. c, Replication kinetics of WT HIV-1 in cells treated with vehicle\n(DMSO) or the RNF114 inhibitor EN219, compared to the replication profile of HIV-1Δasp. d,\nReplication kinetics of WT HIV-1 in cells treated with vehicle or the PHD3 inhibitor Molidustat,\nassessing the effect of PHD3 blockade on viral spreading. Data are presented as means ± SD (n =\n3 donors). *P < 0.05, **P < 0.01, ***P < 0.001 (determined by two-tailed unpaired Student’s t-\ntest at each time point).\n2. Replication readout: Measuring Tat transcript levels by qPCR is not a reliable\nindicator of replication. p24 expression should be quantified by ELISA or flow\ncytometry at several time points.\nWe agree with the reviewer that measuring HIV-1 Tat transcript levels is not a suitable\nproxy for multi-cycle viral spreading. We wish to clarify that our VSV-G pseudotyped\nvirus assays are strictly single-round infections designed to isolate the immediate\ntranscriptional activation following viral entry.\nIn this specific context, quantifying Tat mRNA serves as a rigorous readout for the\nsuccessful initiation of the productive viral life cycle. Crucially, recent quantitative\nanalyses have established that the expression of multiply spliced transcripts (encoding\nTat/Rev) is the most accurate predictor of productive viral gene expression and\ncorrelates strongly with downstream virion production, outperforming other viral\nRNA species5. To ensure terminological precision, we have systematically revised the\nmanuscript. We no longer refer to these single-round results as \"replication.\" Instead,\nwe now explicitly label them as \"viral transcriptional activation\" or \"viral mRNA\nexpression\" throughout the text and figure legends.\nTo address the need for a definitive replication readout, we have performed the\nrequested multi-day time-course experiments using fully replication-competent HIV-1\n(NL4-3 YU2). As detailed in the response to the previous comment regarding the\nreplication assay, we measured viral replication kinetics over a 9-day period. These\nnew data confirm the effects of ASP deletion, mutation, and inhibitor treatment on\nviral propagation in a spreading infection context. By distinguishing between\ntranscriptional readouts (Tat mRNA in single-round assays) and true replication\nkinetics (viral titers in multi-round assays), the revised manuscript now provides a\nprecise and comprehensive assessment of ASP function.\n3. Latency interpretation: ART-treated individuals should not be referred to as having\n“latent infection,” as their viremia is pharmacologically suppressed. Latency\nshould be studied using appropriate models such as GKO or pMorpheus dual\nFluorescent viruses, that allow the discrimination between productive and latent\ninfection.\nWe thank the reviewer for this precise distinction and the constructive suggestion\nregarding experimental models. We apologize for the imprecise terminology in the\noriginal submission. We agree that ART-treated individuals are more accurately\ndescribed as being \"pharmacologically suppressed\" rather than having a \"latent\ninfection\" in the context of our cohort analysis. We have corrected the group labels\nthroughout the manuscript and figures. The groups are now designated as ART-\nsuppressed and Treatment-naive individuals to accurately reflect their clinical status.\nWe appreciate the suggestion by the reviewers regarding the use of dual-fluorescent\nmodels (e.g., GKO or pMorpheus), which are widely recognized as powerful tools for\ninvestigating HIV-1 latency dynamics. However, we respectfully note that previous\nstudies have reported that the HIV-1 antisense transcript (encoding ASP) contributes\nto the maintenance of viral latency by suppressing positive-strand transcription\nthrough epigenetic silencing6. Building upon these foundational observations, the\nprimary objective of our current study is distinct. Our current study focuses on the\ndistinct function of the ASP protein product in the context of active replication.\nTherefore, while we appreciate the suggestion regarding these elegant models and will\nconsider them for future investigations into the latent reservoir, we consider further\ncharacterization of latency mechanisms to be beyond the scope of the current\nmanuscript, which focuses on the replication-enhancing functions of ASP.\n4. Patients’ characterization: Providing additional clinical and immunological\ninformation would greatly enhance the interpretability and reproducibility of the\nresults. Specifically, the authors are encouraged to clarify how the AIDS stage was\ndefined and to include data on CD4⁺ T cell counts, CD4/CD8 ratios, plasma viral load\n(viremia), and any opportunistic infections. Moreover, details regarding antiretroviral\ntherapy (ART)—including treatment duration, current immuno-virological status, and\nthe specific ART regimens or drug classes used—would be highly informative.\nWe appreciate the suggestion from the reviewer to provide a more comprehensive\nclinical profile to enhance the interpretability of our results. To further solidify our\nconclusions, we have collected additional clinical samples, expanding our cohort to\nnow include 17 ART-suppressed individuals and 10 treatment-naive individuals. We\nhave compiled the detailed demographic and immunological data for this cohort into\nExtended Data Table. 2, which now includes patient age, gender, CD4+ T cell counts,\nCD4/CD8 ratios, plasma viral load, duration of ART. This expanded dataset allows for\na more rigorous characterization of the study population.\nBased on these clinical parameters, we categorized the patients into two distinct\ngroups defined as ART Suppressed individuals with pharmacologically suppressed\nviremia, and Treatment Naive individuals with active viremia who have not received\ntherapy. This classification allows us to precisely correlate asp expression levels with\nthe immuno-virological status of the host, ensuring that our comparisons are\nstatistically robust and clinically relevant.\nRegarding the specific details of ART regimens and opportunistic co-infections, we\nwish to be transparent about data availability. Due to the retrospective nature of the\nsample collection and privacy policies at the source clinics, granular records\nconcerning specific drug classes or co-infection events were not accessible for the\nentire cohort. However, we respectfully submit that the available CD4+ counts and\nplasma viral load data provide a definitive and sufficient measure of the patients\nimmuno-virological status, which is the primary variable relevant to the analysis of\nASP expression in this study.\nWe quantified asp transcripts (copies/µg cDNA) referencing the methodology\ndescribed by previous study7, and based on this approach and an expanded sample\nsize, we have revised our results. Consistent with existing literature6, we observed that\nasp RNA is transcribed. Furthermore, previous studies detecting anti-ASP antibodies8\nand specific CD8+ T cell responses9 confirm that this gene product is immunogenic in\nvivo. This finding suggests that the asp gene remains transcriptionally active even\nunder suppressive therapy, providing a reservoir of transcripts that may be rapidly\ntranslated upon viral reactivation or immune modulation.\nIncorporating this improved characterization with our new molecular analysis, we\nobserved that asp RNA is transcribed at substantial levels in both groups (Fig. 1a).\nWhile the detection of asp transcripts in ART-suppressed individuals aligns with\nprevious reports regarding its role in latency maintenance6, the high expression levels\nobserved in treatment-naive, high-viral-load patients suggest a distinct pathological\nfunction. In this context of active infection, consistent with our current findings, the\nfunction of ASP goes beyond solely suppressing sense-strand transcription to actively\ncounteracting host antiviral immunity, thereby facilitating immune evasion and viral\npropagation.\nResponse Figure 6. asp is transcribed at substantial levels in both\nART-suppressed and Treatment-Naive patient cohorts. a, Absolute\nquantification of asp transcript levels (copies per µg cDNA) in\nPBMCs derived from ART-suppressed (n = 17) and Treatment-\nnaive (n = 10) HIV-1 infected individuals. Data are means ± SD.\n*P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s\nt-test).\nmacrophages, which require an R5-tropic virus. This issue needs to be clarified or\nexperimentally corrected.\nWe sincerely thank the reviewer for pointing out this critical detail, and we fully agree\nthat the parental NL4-3 strain is X4-tropic and incapable of efficiently infecting\nmonocyte-derived macrophages. We apologize for the omission in our original\nmanuscript regarding the specific viral construct, and we wish to clarify that we\nexplicitly utilized a macrophage-tropic NL4-3(YU2) chimeric virus rather than the\nparental NL4-3 for all infections using infectious molecular clones in MDMs. This\nmacrophage-tropic clone was generated by replacing the KpnI-to-BamHI fragment\nwithin the env gene of the pNL4-3 backbone with the corresponding sequence from\nthe HIV-1 YU2 isolate, as detailed in established protocols10. Given that the asp gene\nis encoded on the antisense strand of env, we had previously verified via sequence\nalignment that the YU2 isolate (M93258.1) harbors a fully conserved and intact ASP\nopen reading frame, ensuring that the resulting NL4-3 (YU2) chimera retains the\ncapacity to express a functional ASP protein. Consequently, we have corrected the\nMaterials and Methods section and the relevant Figure Legends to explicitly state that\nthe R5-tropic NL4-3(YU2) was used for all MDM infections. In addition, we\nperformed a comparative analysis of p24 release kinetics using infectious molecular\nclones of NL4-3 and NL4-3(YU2) in MDMs, and our data confirmed that the NL4-3\nstrain failed to establish productive infection in these cells, whereas the NL4-3(YU2)\nstrain exhibited robust replication capabilities with a rapid and significant increase in\nviral growth observable as early as one day post-infection.\nRegarding the single-round infection assays in MDMs, we utilized VSV-G\npseudotyped viruses which mediate viral entry via the ubiquitous LDL receptor,\nensuring that these assays are independent of HIV-1 tropism constraints. We apologize\nfor not making this distinction clear in the initial submission, and we have rectified\nthis description throughout the revised manuscript.\nResponse Figure 7. a, NL4-3(YU2) establishes\nproductive infection in MDMs while NL4-3 does not.\nComparative analysis of p24 release kinetics in\nMDMs infected with NL4-3 or NL4-3 (YU2)\ninfectious molecular clones, monitoring viral\nreplication to assess macrophage tropism. Data are\nmeans ± SD (n=3 donors). *P < 0.05, **P < 0.01,\n***P < 0.001 (two-tailed unpaired Student’s t-test).\n6. HIV restriction factors: are specific HIV restriction factors that are ISGs (MX2 for\nexample) upregulated in the absence of ASP? Is phosphorylation of SAMHD1\ndecreased in the presence of ASP?\nWe thank the reviewer for this insightful question regarding the interplay between\nASP and host restriction factors. We focused our investigation on MX2, a potent\ninterferon-stimulated gene (ISG) known to restrict HIV-1 post-entry steps, to\ndetermine if its expression is modulated by ASP.\nTo dynamically track the impact of ASP on restriction factor induction, we infected\nMDMs with wild-type (WT), ASP-deficient (HIV-1Δasp), or P47A-mutant (HIV-1\nP47A) HIV-1 and quantified MX2 mRNA levels at different time points post-infection.\nIn contrast, cells infected with either the HIV-1Δasp or P47A mutants exhibited a\nmarked upregulation of MX2 transcription, particularly at 24 and 48 h (Extended Data\nFig. 4d). This indicates that functional ASP attenuates the transcriptional induction of\nMX2 during infection. To corroborate this finding in primary cells, we re-analyzed our\nRNA-seq dataset derived from HIV-1 infected MDMs. Consistent with mRNA data,\nthe transcriptomic analysis confirmed that MX2 expression levels were significantly\nhigher in the HIV-1Δasp -infected group compared to the wild-type-infected group.\nWe have updated the transcriptomic heatmap in Fig 1h to explicitly include MX2,\nhighlighting its differential regulation by ASP (Fig. 1h). Our observations are\nconsistent with previous conclusions11, 12, indicating that ASP suppresses the\nproduction of IFN, thereby inhibiting the transcription of MX2.\nWe thank the reviewer for the query regarding the impact of ASP on SAMHD1, and to\nrigorously distinguish between the regulation of protein abundance and enzymatic\nactivity, we performed parallel infections using wild-type, HIV-1Δasp, and P47A\nmutant viruses in terminally differentiated MDMs. In the MDMs, the Δasp and P47A\nmutants exhibited only a marginal elevation in SAMHD1 levels at 24 h post-infection\ncompared to the wild-type group, and consistently we did not observe a significant\nreduction in p-SAMHD1 (Thr592) levels as indicated by the densitometric analysis\nlabeled below the figure (Extended Data Fig. 4e), findings that reflect the specific\ncellular context and regulatory mechanisms of macrophages. We attribute the\nobserved molecular patterns to the specific physiology of MDMs. Unlike cycling cells,\nMDMs reside in a G1-like non-proliferating state characterized by low CDK1 activity,\nwhich maintains SAMHD1 in a constitutively dephosphorylated and antivirally active\nform, rendering it unresponsive to further modulation13, 14. Furthermore, the high\nconstitutive expression of SAMHD1 in differentiated macrophages saturates the\nrestriction landscape, limiting the magnitude of interferon-mediated upregulation\ntypically observed in other cell types15, 16.\nIn summary, our combined kinetic profiling and transcriptomic analyses demonstrate\nthat ASP functions as a potent antagonist of the innate immune response. While the\nphenotypic stability of SAMHD1 reflects the cell-cycle constraints intrinsic to MDMs,\nthe pronounced upregulation of MX2 in ASP-deficient mutants confirms that ASP\nprimarily operates by blunting the upstream type I IFN signaling cascade, thereby\npreventing the transcriptional induction of potent restriction factors to facilitate viral\npersistence.\nResponse Figure 8. ASP\ninhibits IFN Signaling to\nsuppress MX2 Transcription.\na, RT-qPCR analysis of MX2\nmRNA levels in MDMs cells\ninfected with VSV-G pseudotyped wild-type, HIV-1Δasp, or HIV-1 P47A viruses, quantified at\nvarious time points post-infection (n=3 donors). b, Immunoblot analysis of SAMHD1 and p-\nSAMHD1 (Thr592) in MDMs infected with VSV-G pseudotyped wild-type, HIV-1Δasp, or HIV-1\nP47A viruses, with densitometric quantification of protein band intensities shown below. Data are\nmeans ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s t-test).\nMinor Comments:\n1. Figure 1• Figure 1A: The label “Latency” is not accurate. These samples represent\npatients on ART with pharmacologically suppressed viremia, which is not equivalent\nto latency. The observed ASP upregulation therefore does not necessarily\nindicate a role in viral persistence.\nWe sincerely thank the reviewer for this accurate critique, and we fully agree that\npatients with pharmacologically suppressed viremia should not be labeled as Latency.\nWe have expanded our clinical cohort and strictly re-categorized the patient groups in\nFigure 1A as ART-Suppressed versus Treatment-Naive to ensure clinical accuracy,\nremoving the Latency designation to prevent over-interpretation of the clinical data.\nScientifically, we have refined our conclusions based on existing literature, and while\nwe acknowledge that ASP has been reported to play a role in maintaining latency6, our\nstudy highlights a novel observation where asp is highly transcribed even in\nTreatment-Naive patients with high viral loads. This pivotal finding prompted us to\ninvestigate the impact of ASP on the host IFN response, leading to our hypothesis that\nhigh ASP expression functions to actively counteract antiviral defenses to facilitate\nviral propagation during active infection rather than solely inducing viral silencing.\nWe sincerely thank the reviewer for this insightful suggestion, and consequently we\nhave excluded the discussion regarding the impact of ASP on viral latency from the\ncurrent manuscript. Instead, building upon established literature and our own novel\nfindings, we have concentrated our focus exclusively on the regulatory mechanism of\nASP against the host IFN response.\n.\n2. Figure 1D: The expression “strongly inhibited” seems overstated. Please either\nmoderate the language or provide quantitative data showing the actual fold reduction\nin phosphorylation induced by ASP.\nWe sincerely appreciate the constructive suggestion of the reviewer to ensure the\nprecision of our descriptive language, and we agree that the term strongly inhibited in\nFigure 1D was subjective. Therefore, we have performed densitometric quantification\non all Western Blot bands assessing phosphorylation levels across the main figures,\nand we have explicitly annotated the relative fold-change values directly below the\ncorresponding bands. Guided by these quantitative results, we have revised the\nmanuscript to moderate our language, and we have replaced the subjective descriptor\nstrongly inhibited with the more precise data-driven term markedly attenuated,\nensuring that our textual descriptions are now strictly aligned with the statistical\nevidence presented in the updated figures.\nResponse Figure 9. a, Immunoblot analysis was\nperformed to assess the phosphorylation status of\nTBK1, IRF3, p38, p65, and ERK in L929 cells\nfollowing HSV-1 stimulation and GFP-ASP\noverexpression.\n3. Figure 1G: The immunofluorescence results are presented qualitatively.\nQuantification of fluorescence intensity or the proportion of positive cells would\nstrengthen the conclusions.\nWe respectfully note that the comment likely refers to Fig. 1e instead of the bubble\nplot in Fig. 1g, and we fully agree that qualitative images are insufficient to rigorously\ndemonstrate IRF3 nuclear translocation. To address this, we performed a quantitative\nanalysis to determine the percentage of cells exhibiting IRF3 nuclear accumulation,\nadopting a scoring method referenced from a recent study17. The resulting quantitative\ndata, now presented as a bar graph in the revised Fig. 1e, clearly demonstrate that ASP\nsignificantly attenuated HSV-1-induced IRF3 nuclear accumulation.\nResponse Figure 10. a,\nImmunofluorescence staining\nvisualizing IRF3 (red) and nuclei\n(DAPI, blue) in HeLa cells\ntransfected with GFP-ASP and\ntreated with PBS or HSV-1 (scale\nbar, 20 μm). Quantification of IRF3 nuclear accumulation (% cells, n = 10 cells per group). Data\nare presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s\nt-test).\n4. Figure 1I: The Western blot for pERK should be replaced with a higher-quality\nimage to allow clearer interpretation.\nWe apologize for the suboptimal quality of the original pERK immunoblot in Figure\n1I which was affected by technical issues during membrane transfer, and we sincerely\nthank the reviewer for pointing this out to ensure the highest standard of data\npresentation. To rectify this, we have repeated the entire experiment and replaced the\nfigure with new high-quality blot images, and importantly these new results are fully\nconsistent with our original findings which confirms the reproducibility of the data\nwhile significantly improving the clarity and interpretability of the figure.\nResponse Figure 11. a, Immunoblot analysis of\nTBK1, IRF3, p38, p65, and ERK phosphorylation in\nMDMs at 24 h post-infection with infectious HIV-1\npNL4-3 (YU2) or pNL4-3 (YU2) Δasp mutant.\n5. Figure 1L: The Western blot suggests that IRF3 nuclear translocation is enhanced\nmainly at 4 h. Please include quantification to support this observation.\nWe appreciate the keen observation of the reviewer regarding the kinetics of IRF3\ntranslocation, and to validate this we performed densitometric quantification of\nnuclear IRF3 levels normalized to the nuclear marker Lamin B1, and consistent with\nthe note of the reviewer our data confirms that while IRF3 nuclear entry is enhanced\nin the HIV-1Δasp group including 4, 8, and 12 h, the difference is indeed most\npronounced at 4 h post-infection. To support this observation directly we have\nexplicitly annotated the relative quantification values below the corresponding bands\nin the revised Figure 1L.\nResponse Figure 12. a, Immunoblot analysis of IRF3 nuclear\ntranslocation in MDMs at different time point post-infection\nwith infectious HIV-1 pNL4-3 (YU2) or pNL4-3 (YU2)\nΔasp mutant.\n6. Figure 2F: The immunofluorescence data should also be quantified rather than\npresented solely as representative images.\nConsistent with our quantitative approach in Fig. 1e, we assessed the protein\ninteraction by determining the percentage of cells exhibiting ASP-TBK1 co-\nlocalization. We analyzed multiple randomized fields of view to calculate the\nproportion of cells showing distinct overlap between ASP and TBK1 signals. The\nresulting quantitative data, now presented as a bar graph in the revised Fig. 2f, clearly\ndemonstrate that HSV-1 infection significantly promoted the co-localization of ASP\nand TBK1.\nResponse Figure 13. a, Confocal microscopy of L929 cells\nexpressing GFP-ASP and FLAG-TBK1, following HSV-1\nstimulation. Scale bars, 50 μm. Quantification of ASP-TBK1 co-\nlocalization (n = 10 cells per group). Data are means ± SD. *P <\n0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired Student’s t-\ntest).\n7. Figure 2G: The Co-IP Western blot appears unconvincing in its current form. A\nclearer, better-resolved blot or additional replicates would strengthen the conclusion.\nWe thank the reviewer for pointing out the need for better image quality, and\nconsequently we have repeated the Co-IP experiment and updated Figure 2G with a\nclearer immunoblot to facilitate interpretation, where the new data remains consistent\nwith our original findings illustrating the physical interaction between ASP and TBK1\nallowing for a clearer visualization of the increased association between these two\nproteins following viral infection.\nResponse Figure 14. a, Co-immunoprecipitation of GFP-ASP\nwith endogenous TBK1 in L929 cells with or without HSV-1\ninfection.\n8. Figure 3N: Please provide Western-Blot to show TBK1 knock-down.\nWe apologize for the oversight in omitting the validation of knockdown efficiency in\nthe original submission, and to address this we have supplemented the manuscript\nwith a Western-Blot confirming that our specific siTBK1 sequence effectively\nsuppresses TBK1 protein expression, and we sincerely appreciate the rigorous\nattention to detail which has helped ensure the completeness of our experimental\nvalidation.\nResponse Figure 15. Validation of TBK1 knockdown efficiency in\nPMs. a, Immunoblot analysis of TBK1 protein expression in PMs\ntransfected with siTBK1, verifying knockdown efficiency.\n9. Figure 6• A higher-quality version of the anti-GFP Western blot would improve\nclarity and the reliability of interpretation.\nWe appreciate the suggestion of the reviewer to improve the presentation of our data,\nand we re-evaluated the anti-GFP blots in Figure 6 acknowledging that high\nbackground levels in the original images compromised their clarity, thus to ensure\nreliability we have repeated the experiments and updated the relevant panels (Fig. 6a-\nc, i-k) with high-quality clear blots that effectively demonstrate the results providing\nsufficient and reliable evidence to substantiate our scientific conclusions.\nResponse Figure 16. a, Immunoprecipitation using hydroxyproline (Hyp) antibody in HEK293T\ncells expressing wild-type ASP or P47A mutant, followed by immunoblot analysis. b,\nImmunoprecipitation using GFP antibody in HEK293T cells expressing wild-type ASP or P47A\nmutant, followed by immunoblot detection of Hyp modification. c, Co-immunoprecipitation in\nHEK293T cells to assess the interaction of ASP with PHD1, PHD2, and PHD3. d, Immunoblot\nanalysis of HEK293T cells co-transfected with wild-type or P47A mutant GFP-ASP and HA-\nPHD3, assessing ASP hydroxylation levels. e, Co-IP assays in HEK293T cells examining the\neffect of PHD3 overexpression on the interaction between ASP and TBK1. f, Co-IP assays in\nHEK293T cells evaluating the influence of PHD3 on the interaction between TBK1 and RNF114\nin the presence of wild-type ASP or ASP P47A.\n10. Figure 7 HIV infection in mice should be followed at different time point showing\nplasma viral load.\nWe sincerely thank the reviewer for this crucial suggestion. In our original design, the\nselection of Day 10 as the endpoint was driven by the specific biological constraints\nof the Hu-PBL mouse model. Unlike HSC-engrafted models, the Hu-PBL model\nrelies on the adoptive transfer of a finite number of mature human PBMCs without\nstem cell renewal. Consequently, the pool of target CD4+ T cells undergoes rapid\ndepletion due to both viral cytopathicity and xenogeneic activation, typically limiting\nthe window of high viremia to approximately 1-2 weeks before target cell exhaustion\noccurs18. Thus, Day 10 was originally chosen to capture the peak viral load before the\ntarget cell pool was depleted.\nWe fully agree that longitudinal monitoring is essential to scientifically validate this\nkinetic model and provide a comprehensive context for our findings. Therefore, we\nestablished a new cohort of Hu-PBL mice to monitor plasma viral loads at Days 5,\n10,\nand 15 post-infection. The results confirmed the expected kinetics showing that WT\nviral loads peaked at Day 10 before declining by Day 15 due to target cell exhaustion,\nand regarding viral genetics we observed that the HIV-1Δasp group exhibited\nsignificantly suppressed viral replication across all time points confirming the\nnecessity of ASP for sustaining infection (Extended Data Fig. 8a), while\npharmacologically the groups treated with EN219 and Molidustat displayed a\ncomparable reduction in viral load validating the translational potential of targeting\nthis pathway (Extended Data Fig. 8b).\nResponse Figure 17. Longitudinal\nmonitoring confirms ASP necessity\nand RNF114-PHD3 targeting efficacy\nin Hu-PBL mice. a, Longitudinal\nanalysis of plasma viral loads in Hu-\nPBL mice infected with wild-type or HIV-1Δasp viruses (NL4-3), measured at Days 5, 10, and 15\npost-infection (n=5 mice per group). b, Longitudinal analysis of plasma viral loads in Hu-\nPBL mice infected with wild-type HIV-1 (NL4-3) and treated with vehicle, EN219, or Molidustat,\nevaluating the therapeutic potential of pharmacological inhibition over a 15-day time course (n=5\nper group). Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed unpaired\nStudent’s t-test).\n11. Line 26: why the authors state “elevated ASP and PHD3 transcript levels are\nnegatively correlated with immune response in ART-treated patients during latency”\nplease clarify.\nWe sincerely appreciate the rigorous critique of the reviewer regarding our statistical\napproach, and we acknowledge that our initial correlation analysis was\nmethodologically inappropriate because ASP represents a variable of emergence while\nhost factors represent variables of magnitude change, thus we have removed this\nanalysis to ensure statistical validity. To strictly evaluate clinical relevance we focused\non the relationship with viral replication specifically within our expanded treatment-\nnaive cohort where we first observed that RNF114 mRNA transcription levels\nexhibited a significant positive correlation with plasma viral load (Fig. 5e, f), and\nconsistent with this pattern we also identified a significant positive correlation\nbetween PHD3 mRNA levels and viral load (Fig. 6f, g), clinical evidence that\nrobustly corroborates our proposed mechanism demonstrating that ASP manipulates\nPHD3 and RNF114 to inhibit IFN production and thereby achieve immune evasion.\nAnd we apologize for the inaccuracies in the original phrasing and analysis and thank\nthe reviewer for pointing this out.\nResponse Figure 18. a, qPCR\nanalysis of RNF114 mRNA levels\nin PBMCs from healthy individuals\n(n = 9), ART-suppressed individuals\n(n = 17), and treatment-naive\nindividuals (n = 10). b, Correlation\nanalysis between RNF114 mRNA\nand plasma viral load (n = 9). one\npatient with viral load > limit of\nquantification was excluded from\ncorrelation analysis. c, qPCR\nanalysis of PHD3 mRNA levels in\nPBMCs from healthy individuals (n\n= 9), ART-suppressed individuals (n\n= 17), and Treatment-Naive individuals (n = 10). d, Correlation analysis between PHD3 mRNA\nand plasma viral load (n = 9). one patient with viral load > limit of quantification was excluded\nfrom correlation analysis. Data are presented as means ± SD, statistical significance was\ndetermined using two-tailed unpaired Student t-test, and correlations were analyzed using Pearson\ncorrelation coefficient for normally distributed data, *P < 0.05 **P < 0.01 ***P < 0.001.\nWe sincerely thank Reviewer 2 for their time and effort in re-evaluating our revised manuscript.\nReference:\n1. Mancarella, A. et al. Detection of antisense protein (ASP) RNA transcripts in individuals\ninfected with human immunodeficiency virus type 1 (HIV-1). Journal of General Virology 100,\n863-876 (2019).\n2. Capoferri, A.A. et al. In vivo detection of HIV-1 antisense transcripts in untreated and ART-\ntreated individuals. bioRxiv (2025).\n3. Waast, L. et al. Strand-specific detection of cell-associated sense and antisense HIV-1 RNAs\nin splenocytes and PBMC from PLWH. Retrovirology (2025).\n4. Zankharia, U. et al. HIV-induced RSAD2/Viperin supports sustained infection of monocyte-\nderived macrophages. J Virol 98, e0086324 (2024).\n5. Zerbato, J.M. et al. Multiply spliced HIV RNA is a predictive measure of virus production ex\nvivo and in vivo following reversal of HIV latency. EBioMedicine 65, 103241 (2021).\n6. Zapata, J.C. et al. The Human Immunodeficiency Virus 1 ASP RNA promotes viral latency by\nrecruiting the Polycomb Repressor Complex 2 and promoting nucleosome assembly. Virology\n506, 34-44 (2017).\n7. Jin, X. et al. APOBEC3G/CEM15 (hA3G) mRNA levels associate inversely with human\nimmunodeficiency virus viremia. J Virol 79, 11513-11516 (2005).\n8. Savoret, J. et al. A Pilot Study of the Humoral Response Against the AntiSense Protein (ASP)\nin HIV-1-Infected Patients. Front Microbiol 11, 20 (2020).\n9. Bet, A. et al. The HIV-1 Antisense Protein (ASP) induces CD8 T cell responses during chronic\ninfection. Retrovirology 12 (2015).\n10. Bobardt, M.D. et al. Contribution of proteoglycans to human immunodeficiency virus type 1\nbrain invasion. J Virol 78, 6567-6584 (2004).\n11. Kane, M. et al. MX2 is an interferon-induced inhibitor of HIV-1 infection. Nature 502, 563-\n566 (2013).\n12. Goujon, C. et al. Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection.\nNature 502, 559-562 (2013).\n13. Badia, R. et al. The G1/S Specific Cyclin D2 Is a Regulator of HIV-1 Restriction in Non-\nproliferating Cells. PLoS Pathog 12, e1005829 (2016).\n14. Cribier, A., Descours, B., Valadao, A.L., Laguette, N. & Benkirane, M. Phosphorylation of\nSAMHD1 by cyclin A2/CDK1 regulates its restriction activity toward HIV-1. Cell Rep 3,\n1036-1043 (2013).\n15. Lahouassa, H. et al. SAMHD1 restricts the replication of human immunodeficiency virus type\n1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol 13, 223-\n228 (2012).\n16. St Gelais, C. et al. SAMHD1 restricts HIV-1 infection in dendritic cells (DCs) by dNTP\ndepletion, but its expression in DCs and primary CD4+ T-lymphocytes cannot be upregulated\nby interferons. Retrovirology 9, 105 (2012).\n17. Han, Z. et al. Palmitoylation of TBK1 enhances the type I interferon signaling and strengthens\nanti-malarial immunity in mice. Nat Commun 16, 10109 (2025).\n18. Brunetti, J.E., Kitsera, M., Munoz-Fontela, C. & Rodriguez, E. Use of Hu-PBL Mice to Study\nPathogenesis of Human-Restricted Viruses. Viruses 15 (2023).\nDear Reviewer,\nThank you for your continued review and favorable assessment of our revised\nmanuscript. We have diligently implemented modifications to address your remaining\nquestions. We are confident that this updated version fully satisfies all raised concerns."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors' revisions have substantially improved the manuscript's scientific rigor. The small epitope-tagged constructs,\nmutational validation, expanded patient cohort, and complete input controls effectively address my major concerns.\nHowever, one issue remains with Figure 1a. The healthy control group present in the original submission has been\nremoved. This negative control is essential to confirm virus-specific asp transcript detection and should be retained to\nestablish assay specificity.", "reviewer_2": "After revision, the manuscript has greatly improved, and I am satisfied with the outcome. The authors have carefully\naddressed all of my previous comments and concerns. They have performed almost all of the requested experiments and\nhave provided clear and reasonable explanations for those that were not conducted. Overall, I am satisfied with the revisions\nand believe the manuscript is now suitable for publication."}, "author_response": "Reviewers Comments\nReviewer #1 (Remarks to the Author):\nThe authors' revisions have substantially improved the manuscript's scientific rigor. The small\nepitope-tagged constructs, mutational validation, expanded patient cohort, and complete input\ncontrols effectively address my major concerns. However, one issue remains with Figure 1a. The\nhealthy control group present in the original submission has been removed. This negative control\nis essential to confirm virus-specific asp transcript detection and should be retained to establish\nassay specificity.\nWe sincerely thank the reviewer for pointing this out. We fully agree that this negative\ncontrol is essential to confirm the virus-specific detection of asp transcripts and\nestablish assay specificity. Accordingly, we have restored the data regarding asp\ncopies from the healthy control group in the revised Figure 1a.\nResponse Figure 1. Quantitative PCR (qPCR) analysis of asp\ntranscript levels in healthy individuals (n = 9 donors), ART-\nsuppressed (n = 17 donors) and treatment-naive (n = 10 donors)\npatient cohorts. Data are means ± SD. *P < 0.05, **P < 0.01,\n***P < 0.001 (one-way ANOVA followed by Tukey’s multiple\ncomparisons test).\nReviewer #2 (Remarks to the Author):\nAfter revision, the manuscript has greatly improved, and I am satisfied with the\noutcome. The authors have carefully addressed all of my previous comments and\nconcerns. They have performed almost all of the requested experiments and have\nprovided clear and reasonable explanations for those that were not conducted. Overall,\nI am satisfied with the revisions and believe the manuscript is now suitable for\npublication.\nWe sincerely thank the reviewer for their continued guidance, constructive\nsuggestions, and ultimate recommendation for publication. We are delighted that our\ncomprehensive revisions and explanations have fully resolved your concerns to your\nsatisfaction."}]

Decision letter