ZFP36L2 is an interferon β -induced inhibitor that restricts the nuclear export of HIV-1 transcripts

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
Type I interferons restrict HIV-1 replication by inducing antiviral genes, but the full spectrum of their effectors remains incompletely defined. Here we identify ZFP36L2, a nuclear RNA-binding protein, as an IFN-β-induced inhibitor of HIV-1 infection. Silencing of ZFP36L2 impairs IFN-β-mediated HIV-1 inhibition, whereas overexpression of ZFP36L2 suppresses viral replication. Notably, reconstitution of ZFP36L2 in CD4⁺ T cells from HIV-1-infected individuals reduces viral spread ex vivo, and ZFP36L2 transcript levels inversely correlate with plasma viral loads in vivo. Mechanistically, ZFP36L2 binds to the HIV-1 Rev protein and inhibits the nuclear export of Rev response element-containing viral transcripts, thereby blocking downstream viral protein expression. A Rev mutant lacking amino acids 109–116 fails to bind ZFP36L2 and exhibits resistance to ZFP36L2-mediated inhibition, underscoring the functional significance of this interaction. These findings establish ZFP36L2 as an IFN-β-induced antiviral factor that suppresses HIV-1 replication through Rev-dependent inhibition of viral RNA export.

Introduction
Human immunodeficiency virus (HIV-1) infection in humans induces innate immune responses that are primarily mediated by type I interferons (IFN-Is), which are antiviral cytokines comprising 12 different IFN-α subtypes and IFN-β. They exert broad antiviral effects by activating interferon-stimulated genes (ISGs) 1-9. Upon HIV-1 infection, pattern recognition receptors and cytosolic sensors in human immune cells detect viral cDNA and RNA. Viral cDNA is first recognized by IFN-inducible protein 16 (IFI16) or cyclic GMP–AMP (cGAMP) synthase, triggering IFN-I production via IFI16 and the cGAMP-activating stimulator of the IFN gene (STING) 10-13. Extensive in vitro studies have identified several ISGs, such as IFN-induced transmembrane proteins (IFITMs), APOBEC3G, Tetherin, SAMHD1, MX2, and Shiftless that exert potent anti-HIV-1 activity 8, 9, 14-23. These findings suggest that IFN-I responses triggered by HIV-1 infection play a crucial role in limiting viral spread.

HIV-1 gene expression is regulated by several posttranscriptional mechanisms, including RNA splicing, stability, transport, and translation. Because the HIV-1 genome contains an AU-rich sequence, rapid nuclear export facilitated by the Rev protein is necessary to prevent excessive splicing. The HIV-1 Rev response element (RRE), a ~350 bp, highly structured RNA element located within the env-coding region of the viral genome, is essential for viral replication and is highly conserved across different HIV-1 isolates. During the late phase of the viral life cycle, viral proteins are translated from unspliced (9 kb) and partially spliced (4 kb) RRE-containing RNAs. These intron-containing RNAs are typically retained in the nucleus for splicing or degradation. However, their nuclear export is achieved through the cooperative binding of multiple Rev molecules to the RRE. This assembly recruits the host Crm1/Ran-GTP nuclear export machinery via the nuclear export signal of Rev. Once in the cytoplasm, these RNAs are either translated into viral proteins or packaged as genomic RNA for new virions. Notably, HIV-1 infection occurs in activated CD4+ T cells, where cytokine and chemokine mRNA decay is regulated by the binding of AU-rich elements to the mRNA-destabilizing protein ZFP36 (tristetraprolin). ZFP36 belongs to a family of CCCH zinc finger-containing RNA-binding proteins, which includes ZFP36L1 and ZFP36L2 24, 25. These human ZFP36 family proteins contain two conserved zinc finger domains and typically bind to adenine-uridine (AU)-rich elements (AREs) in the 3′ untranslated regions (3′-UTR) of cytokine mRNAs, exerting anti-inflammatory activity via transcript destabilization 25-30. Moreover, ZFP36 antagonizes Myc-induced lymphomagenesis 31, while its paralogs, ZFP36L1 and ZFP36L2, play redundant roles in preventing T cell leukemia in mice and promoting cell quiescence 32, 33. Furthermore, ZFP36 directly inhibits HIV-1 production by binding to genomic RNA 34, while ZFP36L1 inhibits influenza A virus replication through translational repression by selectively targeting viral RNA 35.

In this study, we identified ZFP36L2 as a downstream effector of IFN-β but not IFN-α that mediates the inhibition of HIV-1 infection. Our findings reveal that IFN-β significantly induces ZFP36L2 expression in CD4+ T lymphocytes and macrophages. Notably, ZFP36L2 overexpression reduces susceptibility to multiple lentiviruses, including HIV-1, HIV-2, simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV), but not to feline immunodeficiency virus (FIV) or murine leukemia virus (MLV). In addition, ZFP36L2 overexpression restricts ex vivo HIV-1 spread in CD4+ T cells from HIV-1-infected individuals. In untreated HIV-1-infected individuals, ZFP36L2 transcript levels in CD4+ T cells inversely correlated with viral loads. Conversely, ZFP36L2 depletion via RNA interference (RNAi) diminished the anti-HIV-1 activity of IFN-β in CD4+ T cells and macrophages. Mechanistic studies revealed that ZFP36L2 inhibits the nuclear export of RRE-containing viral transcripts by interacting with the HIV-1 Rev protein. Therefore, ZFP36L2 is a critical effector of the anti–HIV-1 activity mediated by type I IFN-β, shedding light on the intricate molecular mechanisms underlying the IFN-β–driven antiviral response against HIV-1.

Results
ZFP36L2 restricts HIV-1 infection
To investigate the potential anti-HIV activity of the ZFP36 family—given that ZFP36 inhibits HIV-1 production by binding to viral genomic RNA—we first overexpressed its family members, ZFP36, ZFP36L1 and ZFP36L2, along with the proviral vector HIV-1 NL4-3 in 293T cells and assessed viral production. All members of the ZFP36 family could inhibit HIV-1 titers (Supplementary Fig. 1a) when expressed at comparable protein levels, with ZFP36L2 exhibiting the most potent inhibitory effect. Subsequently, we further investigated the anti-HIV activity of ZFP36L2 by coexpressing it with the HIV-1 NL4-3 proviral vector in 293T cells. Viral titers were significantly reduced in a dose-dependent manner, with a 15- to 41.5-fold decrease with increasing ZFP36L2 expression levels (Fig. 1a,b). Notably, Gag and Env levels were reduced, whereas Nef levels remained unaffected regardless of ZFP36L2 expression. These findings suggest that ZFP36L2 inhibits the production of HIV-1 progeny virions in producer cells without altering Nef protein levels. Next, we assessed ZFP36L2 against other HIV-1 isolates, including the CCR5-tropic BaL and AD8, and dual-tropic 89.6. In all cases, p24 levels were decreased in the presence of ZFP36L2 (Fig. 1c), indicating that ZFP36L2 inhibits the production of progeny virions across these HIV-1 strains. Moreover, exogenous ZFP36L2 consistently inhibited HIV-1 progeny virion production in stimulated CD4+ T lymphocytes (Fig. 1d).

To determine whether ZFP36L2 could restrict HIV-1 spread in T cells, we generated ZFP36L2-expressing Jurkat T cells using lentiviral vectors. HIV-1 spread was significantly inhibited in ZFP36L2-expressing Jurkat T cells compared to GFP-expressing control cells (Supplementary Fig. 1c–e). Notably, ZFP36L2 expression did not evidently affect cell proliferation (Supplementary Fig. 1c), indicating that the observed inhibitory effect was due to ZFP36L2 and not cell toxicity. Similarly, in stimulated primary CD4+ T lymphocytes, ZFP36L2 effectively restricted viral spread (Fig. 1e–g). To further substantiate these findings, we examined the effect of ZFP36L2 on HIV-1 spread in primary macrophages. Consequently, ZFP36L2 inhibited the spread of CCR5-tropic HIV-1 in nondividing primary macrophages (Fig. 1h,i). Furthermore, we also examined lactate dehydrogenase (LDH) release as a measure of toxicity and found no significant toxicity associated with ZFP36L2 expression (Fig. 1j), thereby confirming that the observed inhibition of HIV-1 proliferation in the presence of ZFP36L2 is not due to cell toxicity.

Next, we determined whether ZFP36L2 exhibits broad antiviral activity. We challenged ZFP36L2-overexpressing cells with HIV-2, SIV, FIV, MLV, and HBV. Our findings demonstrated that ZFP36L2 inhibited HIV-1, HIV-2, and SIV, but had no significant effect on FIV, MLV, or HBV. Specifically, ZFP36L2 strongly inhibited the titers of HIV-2 (Fig. 1k), SIV (Fig. 1l), and EIAV (Fig. 1m) in producer cells. However, no significant inhibitory effect was observed for FIV, MLV, or HBV titers (Fig. 1n–p). These findings suggest that ZFP36L2 preferentially restricts primate lentiviruses and does not exhibit broad-spectrum antiviral activity against retroviruses. The mechanism underlying the resistance of FIV and MLV to ZFP36L2-mediated inhibition requires further investigation.

IFN- induces ZFP36L2 expression
We assessed endogenous ZFP36L2 expression across multiple cell types and found it to be constitutively expressed in primary macrophages, monocytes, and PMA-stimulated THP-1 cells. Lower levels of ZFP36L2 were detectable in stimulated CD4⁺ T lymphocytes and dendritic cells, whereas ZFP36L2 expression was undetectable in 293T, HeLa, and Jurkat cell lines (Supplementary Fig. 2a). To investigate interferon regulation, stimulated CD4⁺ T cells were treated with IFN-α, IFN-β, or IFN-γ. Among these, only IFN-β robustly induced ZFP36L2 expression (Supplementary Fig. 2b,c). A similar trend was observed in primary macrophages, where IFN-β further enhanced the already high basal ZFP36L2 levels. IFN-γ induced a modest upregulation, while IFN-α had no significant effect on ZFP36L2 expression (Supplementary Fig. 2d,e). We next examined whether other members of the ZFP36 family are responsive to interferon signaling. In both stimulated CD4⁺ T cells and macrophages, only ZFP36L2—not ZFP36 or ZFP36L1—was significantly upregulated by IFN-β (Supplementary Fig. 2f,g), suggesting ZFP36L2 as the primary IFN-β-responsive member in these cells. Consistently, among the ZFP36 family proteins, ZFP36L2 exhibited the strongest anti-HIV-1 activity when overexpressed (Supplementary Fig. 1a,b), suggesting its distinct role in IFN-β-mediated antiviral defense.

To compare ZFP36L2 regulation with that of MX2, a well-established IFN-I-induced HIV-1 restriction factor, we analyzed the expression kinetics of both genes in response to interferon stimulation. While IFN-α and IFN-β strongly induced MX2 in CD4⁺ T cells and macrophages, only IFN-β significantly upregulated ZFP36L2 (Supplementary Fig. 2h,i). ZFP36L2 and MX2 showed distinct induction profiles: MX2 peaked at 12 h and declined thereafter, whereas ZFP36L2 expression gradually increased, peaking at 48 h poststimulation. These kinetic differences were leveraged in subsequent IFN-β experiments to specifically study ZFP36L2-mediated antiviral activity.

ZFP36L2 Inhibits the nuclear export of RRE-containing HIV-1 transcripts
To investigate the molecular mechanism by which ZFP36L2 inhibits HIV-1 production, we first examined whether it affects the early stages of the viral replication cycle. Using HIV-1 NL4-3.Luc. R⁻.E⁻ reporter vectors, we found that ZFP36L2 did not reduce luciferase activity in producer cells (Fig. 2a). Next, we separated nuclear and cytoplasmic fractions from 293T cells. Although total Gag RNA levels remained unchanged, cytoplasmic Gag RNA levels were markedly reduced (by approximately 11.9-fold), while nuclear Gag RNA levels increased (by approximately 2.1-fold) in the presence of ZFP36L2 (Fig. 2b). These changes are consistent with a model in which ZFP36L2 impairs the nuclear export of HIV-1 Gag RNA. This result supports the conclusion that ZFP36L2 blocks the nuclear export of Gag RNA, leading to its nuclear accumulation. The separation of nuclear and cytoplasmic fractions was validated using the following protein markers— Lamin B (nuclear), GAPDH (cytoplasmic), and p24Gag (viral) (Fig. 2c, left) —as well as the nuclear RNA marker U6 RNA (Fig. 2c, right). Moreover, reverse transcription-negative RT-qPCR did not yield any amplification, confirming the absence of contamination from proviral DNA (Fig. 2b). These findings suggest that ZFP36L2 restricts the cytoplasmic transport of Gag RNA, promoting its nuclear retention. Consistent effects were observed in Jurkat T cells (Supplementary Fig. 3a–c) and CD4⁺ T lymphocytes (Supplementary Fig. 3d–f), where ZFP36L2 similarly reduced cytoplasmic and increased nuclear Gag RNA levels. To determine whether ZFP36L2 directly targets Gag RNA, we cloned the Gag sequence into an exogenous expression vector and cotransfected it with ZFP36L2 into 293T cells. In this context, ZFP36L2 did not affect the nuclear export of exogenous Gag RNA (Supplementary Fig. 3g), indicating that its activity requires specific HIV-1 cis-acting elements.

We then analyzed different HIV-1 transcript classes derived from the wild-type HIV-1 NL4-3 proviral vector (Fig. 2d) in the presence or absence of ZFP36L2. In the cytoplasm, ZFP36L2 selectively reduced unspliced (gag-pol) and singly spliced (vif, vpr, vpu/env) transcript levels, whereas those of multiply spliced transcripts (tat, rev, nef) remained unaffected (Fig. 2e). Conversely, in the nucleus, gag-pol and singly spliced transcript levels increased, whereas those of multiply spliced transcripts remained unchanged. The total RNA levels of all transcript classes (gag-pol, vif, vpr, vpu/env, tat, rev, and nef) were unaffected, indicating that ZFP36L2 selectively inhibits the nuclear export of unspliced and singly spliced transcripts without altering their synthesis or stability. To validate these findings, we performed RT-PCR followed by Southern blotting using primers (forward primer FP; reverse primers RP1 and RP2, as indicated) that amplify all singly and multiply spliced HIV-1 transcripts (Fig. 2f). Consistently, ZFP36L2 expression significantly reduced cytoplasmic levels and increased nuclear levels of singly spliced transcripts, whereas multiply spliced transcripts remained unaffected in both compartments (Fig. 2g). Proper cellular fractionation was confirmed by analyzing compartment-specific protein markers and U6 snRNA (Fig. 2h,i). Notably, the HIV-1 RRE is present in unspliced and singly spliced transcripts but absent from multiply spliced transcripts (Fig. 2d), suggesting that ZFP36L2 specifically targets and inhibits the nuclear export of RRE-containing transcripts, thereby interfering with HIV-1 replication.

ZFP36L2 interacts with HIV-1 transcripts via Rev binding
To determine whether ZFP36L2 requires the RRE to inhibit nuclear export, we used a gag-pol expression vector with or without RRE (Fig. 3a). In the absence of RRE, ZFP36L2 failed to inhibit cytoplasmic viral RNA accumulation, regardless of whether Rev was coexpressed (Fig. 3b,c). However, when RRE was present, ZFP36L2 restored its inhibitory effect, but only in the presence of rev (Fig. 3d,e). Under these conditions, ZFP36L2 reduced cytoplasmic gag RNA levels by ~11-fold and increased nuclear gag RNA levels by ~1.9-fold, without altering total gag RNA levels—consistent with impaired nuclear export. Proper cellular fractionation was validated using Lamin B (nuclear), GAPDH (cytoplasmic), rev, and ZFP36L2 as markers (Fig. 3c,e). Moreover, the absence of proviral DNA contamination was confirmed by RT-minus qPCR controls. These results suggest that ZFP36L2 does not directly recognize the RRE directly but instead acts through a Rev-dependent mechanism.

To investigate whether ZFP36L2 physically interacts with Rev, we first evaluated its ability to bind HIV-1 Rev versus FIV Rev, since ZFP36L2 inhibits HIV-1 but not FIV. Co-immunoprecipitation assays demonstrated that ZFP36L2 interacts with HIV-1 Rev but not FIV Rev (Supplementary Fig. 4a–c). Subsequently, we expanded this analysis to Rev proteins from HIV-2, SIVmac239, and EIAV. ZFP36L2 was found to bind to Rev from HIV-1, HIV-2, SIVmac239, and EIAV, but not FIV (Supplementary Fig. 4d), correlating with its antiviral activity against these viruses. Notably, ZFP36L2 did not inhibit MLV or HBV, which lack a Rev homolog, further supporting a Rev-dependent mechanism. To determine whether the interaction between ZFP36L2 and Rev is RNA-mediated, we performed co-immunoprecipitation in the presence of nuclease. The interaction persisted (Fig. 3f, left and right), indicating that it is RNA-independent and likely reflects direct protein–protein binding. This was further confirmed using in vitro pulldown assays with recombinant His-tagged ZFP36L2 protein, which bound directly to HIV-1 and EIAV Rev, but not FIV Rev (Supplementary Fig. 4e,f). Because all ZFP36 family members exhibit some degree of anti-HIV activity, we assessed whether ZFP36 and ZFP36L1 also bind Rev. Both proteins bound HIV-1 Rev, although more weakly than ZFP36L2 (Supplementary Fig. 4g). This weaker binding reflects their relatively modest inhibitory effects on HIV-1 production compared to that of ZFP36L2 (Supplementary Fig. 1a,b), highlighting the superior affinity and functional potency of ZFP36L2 among the family members.

To determine the importance of the ZFP36L2–Rev interaction in antiviral function, we generated GFP-tagged ZFP36L2 deletion mutants (Supplementary Fig. 5a). Deletion of the C-terminal region (amino acids 446–480) abrogated binding to Rev (Supplementary Fig. 5b, left and right), severely impairing anti-HIV-1 activity (Supplementary Fig. 5c,d). In contrast, mutants that retained Rev binding maintained their antiviral activity. These findings establish that binding to Rev is essential for the ZFP36L2-mediated inhibition of HIV-1 replication. Finally, we examined the antiviral activity of ZFP36L2 orthologs from human, mouse, rhesus macaque, and Felis catus against HIV-1, HIV-2, SIV, FIV, and MLV. ZFP36L2 from human, mouse, and macaque—but not from Felis catus—successfully inhibited HIV-1 and SIV production (Supplementary Fig. 6a,b). None of the orthologs inhibited FIV or MLV (Supplementary Fig. 6c,d). Binding assays showed that only human, mouse, and macaque ZFP36L2 bound to HIV-1 and SIV Rev (Supplementary Fig. 6e,f), while none interacted with FIV Rev (Supplementary Fig. 6g). These results account for the observed species-specific antiviral activity and support the conclusion that ZFP36L2 selectively inhibits Rev-dependent lentiviruses by binding to rev and blocking the nuclear export of RRE-containing viral transcripts.

HIV-1rev (109-116) mutant is resistant to ZFP36L2-mediated inhibition
To further substantiate the role of Rev in ZFP36L2-mediated inhibition, we generated a panel of HIV-1 Rev deletion mutants (Δ27–50, Δ51–72, Δ73–91, Δ91–99, Δ100–108, and Δ109–116) to identify regions required for interaction with ZFP36L2. Co-immunoprecipitation assays revealed that Δ73–91 and Δ109–116 Rev mutants failed to bind ZFP36L2, despite exhibiting comparable ZFP36L2 pulldown efficiency (Supplementary Fig. 7a, left and right). To assess functional competence, we tested whether these mutants could replace wild-type Rev in rescuing Rev-defective HIV-1 production. Only the Δ109–116 mutant Rev supported virus production at levels similar to those of wild-type Rev, whereas the Δ73–91 mutant was nonfunctional (Supplementary Fig. 7b). These results suggest that the Δ109–116 mutant may retain functional activity but evades ZFP36L2 inhibition. To directly investigate this, we expressed either wild-type or Δ109–116 mutant Rev in the context of Rev-defective HIV-1 and evaluated viral production with or without ZFP36L2. As expected, viral production was not observed without Rev (Fig. 3g). Both wild-type and Δ109–116 mutant Rev rescued HIV-1 production comparably; however, ZFP36L2 inhibited only wild-type Rev-mediated production. In contrast, ZFP36L2 had no significant effect on viral output in the presence of the Δ109–116 Rev mutant. We next examined viral RNA localization. ZFP36L2 inhibited cytoplasmic accumulation and promoted nuclear retention of viral transcripts in the presence of wild-type Rev, but not Δ109–116 mutant Rev (Fig. 3h). The fractionation efficiency was validated by assessing GAPDH and Lamin B levels. Similarly, ZFP36L2 suppressed Gag protein expression only when wild-type Rev was present (Fig. 3i), further confirming that it blocks Rev-mediated nuclear export of viral RNA.

To confirm these findings in the context of replication-competent HIV-1, we replaced the rev gene in the HIV-1 proviral vector with the Δ109–116 mutant and assessed the effect of ZFP36L2. ZFP36L2 could no longer inhibit HIV-1revΔ109–116 production in HeLa cells, in contrast to its potent suppression of wild-type virus (Fig. 4a). Similar resistance was observed in Jurkat T cells (Fig. 4b,c) and primary CD4⁺ T lymphocytes (Fig. 4d,e), confirming that the Δ109–116 mutation renders HIV-1 resistant to ZFP36L2 inhibition. To evaluate whether ZFP36L2 affects the spread of HIV-1revΔ(109-116), we monitored replication kinetics over 8 days. Although ZFP36L2 suppressed the replication of wild-type HIV-1 in both Jurkat cells (Fig. 4f,g) and CD4⁺ T cells (Fig. 4h,i), HIV-1revΔ109–116 replication remained unaffected. Importantly, ZFP36L2 expression did not impair host cell proliferation (Supplementary Fig. 1c), excluding cell growth effects as a confounding variable. Notably, the HIV-1revΔ109–116 mutant exhibited reduced replication ability compared to wild-type HIV-1, likely owing to the partial functional deficiency of mutant Rev, as reflected by its lower Gag production in producer cells (Fig. 4a,b,d).

To investigate the molecular basis of this resistance, we assessed whether ZFP36L2 could still associate with viral RNA in the presence of mutant Rev. RNA immunoprecipitation revealed that ZFP36L2 bound to Gag transcripts in wild-type HIV-1, but not in the HIV-1revΔ109–116 context (Supplementary Fig. 7c,d). RT-minus qPCR confirmed that these results were not due to DNA contamination. Together, these findings demonstrate that ZFP36L2 requires direct interaction with Rev to bind viral RNA and inhibit its nuclear export. The Δ109–116 Rev mutant, which cannot bind ZFP36L2, functions as an escape mutant that confers resistance to ZFP36L2-mediated inhibition.

IFN- induces ZFP36L2 expression to inhibit HIV-1 infection
To determine whether endogenous ZFP36L2 restricts HIV-1 replication, we first evaluated its function in primary macrophages, where it is constitutively expressed. RNA interference (RNAi)-mediated ZFP36L2 knockdown in macrophages infected with CCR5-tropic HIV-1 AD8 resulted in significantly enhanced viral spread over a 15-day period (Supplementary Fig. 8a,b), indicating that ZFP36L2 intrinsically limits HIV-1 infection in these cells. Moreover, endogenous ZFP36L2 consistently restricted HIV-1 Gag, but not Rev, RNA nuclear export in macrophages (Supplementary Fig. 8c–e). Given that IFN-β, but not IFN-α, upregulates ZFP36L2 expression in both CD4⁺ T cells and macrophages, we investigated whether IFN-β-mediated HIV-1 inhibition is dependent on ZFP36L2. Notably, IFN-β also induces the expression of the host restriction factor MX2. To specifically evaluate the contribution of ZFP36L2, we exploited the differential expression kinetics of these two genes: ZFP36L2 expression peaks at 48 h posttreatment, whereas MX2 levels decline (Supplementary Fig. 2h,i). Accordingly, CD4⁺ T cells and macrophages were treated with IFN-β for 48 h before HIV-1 infection. In CD4⁺ T cells, ZFP36L2 knockdown markedly impaired IFN-β-mediated suppression of HIV-1 production, while IFN-α-mediated inhibition remained unaffected (Fig. 5a,b). In control cells, IFN-β reduced viral titers by 7.2- and 7.7-fold, whereas in ZFP36L2-depleted cells, suppression was reduced to only 1.3- and 1.2-fold. In contrast, IFN-α reduced HIV-1 titers to a similar extent in both control and ZFP36L2 knockdown cells (6.1- vs. 5.4-fold; 6.6- vs. 5.9-fold). Immunoblotting confirmed effective ZFP36L2 silencing and showed that ZFP36L2 expression was induced by IFN-β but not IFN-α. Correspondingly, Gag protein levels were restored in IFN-β-treated ZFP36L2-deficient cells, but not in IFN-α-treated cells. The modest increase in viral titers following ZFP36L2 knockdown under unstimulated conditions suggests that basal ZFP36L2 partially restricts HIV-1 in CD4⁺ T cells. Similar ZFP36L2-dependent IFN-β inhibition was observed in primary macrophages (Supplementary Fig. 8f,g). ZFP36L2 silencing impaired the ability of IFN-β—but not IFN-α—to suppress HIV-1. Because of the higher basal ZFP36L2 levels in macrophages (Supplementary Fig. 2a), knockdown alone increased viral replication, even without IFN stimulation.

MX2 primarily acts during early replication by blocking viral core translocation from the cytoplasm to the nucleus, whereas ZFP36L2 exerts inhibitory effects at later stages. To minimize the antiviral contribution of MX2, we electroporated proviral HIV-1 NL4-3.Luc. R-.E- vectors into CD4⁺ T cells in the presence or absence of IFNs. In these transfected cells, HIV-1 completes only the late stages of replication, during which MX2 has no effect, because it acts early in the viral life cycle. This approach allowed us to specifically assess the role of ZFP36L2. Under these conditions, ZFP36L2 knockdown continued to impair IFN-β-mediated suppression of HIV-1 titers in CD4⁺ T cells (Supplementary Fig. 9a,b), suggesting that IFN-β induces ZFP36L2 to function in producer cells, where it inhibits HIV-1 production.

To further confirm the role of ZFP36L2 in IFN-β-mediated HIV-1 inhibition, we evaluated infections with wild-type or rev(109-116) mutant HIV-1, which is resistant to ZFP36L2, in CD4⁺ T cells treated with or without IFNs (Fig. 5c,d). IFN-α and IFN-β reduced wild-type HIV-1 titers by 15.2- and 12.2-fold (Fig. 5c) and 11.3- and 9.03-fold (Fig. 5d), respectively. In contrast, rev(109-116) mutant HIV-1 titers were reduced by 13.1- and 10.7-fold with IFN-α, but only by 1.45- and 2.1-fold with IFN-β. This suggests that the mutant is resistant to ZFP36L2-mediated IFN-β inhibition, but remains sensitive to IFN-α, likely because of mechanisms independent of ZFP36L2. To confirm this finding, we knocked down ZFP36L2 in HIV-1-infected CD4⁺ T cells and assessed the effects of IFN stimulation. In wild-type HIV-1 infections, ZFP36L2 knockdown reduced IFN-β-mediated inhibition from 7.2-fold to 1.3-fold (Fig. 5e), confirming the role of ZFP36L2 in this suppression. However, IFN-α inhibition remained unchanged (6.1- vs. 5.4-fold). In rev(109-116) mutant HIV-1 infections, IFN-β conferred only a modest 1.6-fold reduction in viral titers, which was unaffected by ZFP36L2 knockdown (1.6- vs. 1.2-fold), indicating that IFN-β acts via ZFP36L2-independent pathways. Immunoblotting confirmed successful ZFP36L2 depletion and revealed that Gag protein levels were restored in wild-type—but not rev(109-116)—infected cells treated with IFN-β (Fig. 5f). Similar results were observed in primary macrophages transfected with proviral vectors encoding wild-type or rev(109-116) mutant HIV-1, with or without IFN treatment (Supplementary Fig. 9c,d). Importantly, this proviral transfection approach reduces the influence of endogenous MX2, which acts in the early viral life cycle stage but has no effect in producer cells. Consistently, ZFP36L2 depletion impaired IFN-β-mediated suppression of wild-type HIV-1, but not rev(109-116) HIV-1 titers. Notably, macrophages expressed higher basal levels of ZFP36L2 than CD4⁺ T cells even without IFN stimulation. Overall, these findings demonstrate that IFN-β induces ZFP36L2 to inhibit HIV-1 replication in both CD4⁺ T cells and macrophages, and that this effect is circumvented by the rev(109-116) mutant, which is resistant to ZFP36L2-mediated inhibition.

IFN- induces ZFP36L2 expression to inhibit HIV-1 spread in CD4+ T cells from HIV-1-infected individuals ex vivo
To investigate the role of ZFP36L2 in HIV-1 infection in vivo, we first analyzed ZFP36L2 mRNA levels in CD4⁺ T cells from three groups: ART-naive HIV-1-infected individuals (Supplementary Table 1), ART-treated individuals with suppressed viral loads (<50 copies/mL; Supplementary Table 2), and healthy donors. ZFP36L2 expression was comparable between healthy donors and ART-treated individuals but was significantly increased in CD4⁺ T cells from ART-naive individuals (Fig. 6a). This upregulation may result from endogenous IFN or cytokine stimulation during active HIV-1 infection. To further assess the anti-HIV-1 function of ZFP36L2 in vivo, we analyzed correlations between ZFP36L2 expression in CD4⁺ T cells and its clinical parameters. ZFP36L2 transcript levels were inversely correlated with plasma HIV-1 RNA levels (Fig. 6b). This correlation may not be specific, as ZFP36L2 is an IFN-stimulated gene, and similar correlations may also be observed for other IFN-induced genes. Moreover, ZFP36L2 expression was inversely correlated with CD4⁺ T cell counts (Fig. 6c); however, it exhibited no correlation with the CD4⁺/CD8⁺ T cell ratio (Fig. 6d), suggesting that ZFP36L2 activity is closely associated with HIV-1-infected CD4⁺ T cells and may contribute to CD4⁺ T cell depletion.

To directly assess the antiviral function of ZFP36L2 ex vivo, we isolated total CD4⁺ T cells from ART-naive and ART-treated HIV-1-infected individuals with suppressed viral loads (<50 copies/mL; their backgrounds in Supplementary Table 3). Following stimulation for lentiviral transduction, cells were transduced with either a wild-type or Δ446–480 mutant ZFP36L2-GFP expression vector or a GFP-only control vector and subsequently subjected to puromycin selection (Fig. 6e). Exogenous ZFP36L2 expression did not affect CD4⁺ T cell proliferation (Fig. 6f), however, it significantly inhibited HIV-1 spread compared to the Δ446–480 mutant ZFP36L2-GFP or the GFP-only control (Fig. 6g,h). These results demonstrate that ZFP36L2 overexpression suppresses HIV-1 replication in CD4⁺ T cells ex vivo and highlight its potential as a gene therapy candidate. We next investigated whether IFN-β could induce ZFP36L2 to inhibit HIV-1 replication in CD4⁺ T cells ex vivo. CD4⁺ T cells isolated from ART-naive HIV-1-infected individuals (Supplementary Table 4) were transduced with lentiviral vectors encoding either control or ZFP36L2-specific shRNA in the presence or absence of IFN-β. ZFP36L2 knockdown significantly impaired the ability of IFN-β to suppress HIV-1 production (Fig. 6i). Moreover, virion production was inversely correlated with ZFP36L2 expression levels (Fig. 6j). Cell proliferation was unaffected by ZFP36L2 knockdown (Fig. 6k), indicating that the reduction in IFN-β-mediated suppression was due to ZFP36L2 depletion. Similar results were observed in CD4⁺ T cells obtained from ART-treated HIV-1-infected individuals: ZFP36L2 knockdown impaired IFN-β-mediated inhibition of HIV-1 spread ex vivo (Fig. 6l–n). Taken together, these findings demonstrate that IFN-β induces ZFP36L2 expression to impede HIV-1 replication in CD4⁺ T cells ex vivo, validating the physiological relevance of this antiviral pathway in human HIV-1 infection.

Methods
Ethics statement
This study (Protocol No. 2021-328-2 on June 24 2021) is approved by the Research and Ethics Committee of The First Hospital of China Medical University. All blood samples were obtained from healthy donors following National Health and Medical Research Council guidelines. In addition, informed consent was obtained from each healthy donor prior to the study.

Biosafety statement
All experiments involving infectious materials were conducted in a certified Biosafety Level 3 (BSL-3) facility in accordance with institutional and national guidelines. These protocols ensured the safe conduct of research and the protection of personnel and the environment. This study advances the fundamental understanding of host antiviral defenses and may have potential therapeutic implications. With regard to potential Dual-Use Research of Concern (DURC) associated with the HIV-1 RevΔ(109–116) mutant described in this study, this mutant virus was generated solely for mechanistic investigations and was examined exclusively in controlled in vitro and ex vivo experimental systems. Importantly, the mutant does not exhibit enhanced replication capacity or pathogenicity compared with the wild-type virus; rather, it demonstrates reduced replication capacity relative to wild-type HIV-1. All experiments were conducted in strict compliance with institutional biosafety regulations and were reviewed and approved by the appropriate biosafety oversight committees.

HIV-1-infected individuals
ART-naive or treated patients with viral loads of <50 copies/mL and untreated HIV-1+ patients were enrolled in this study. Peripheral blood mononuclear cells (PBMCs) from patients were prepared using Ficoll-Hypaque density gradient centrifugation. CD4+ T cells were isolated from PBMCs by negative selection using a human CD4+ T-cell enrichment cocktail (Stem Cell Technologies). Ethical approval was obtained from the Research and Ethics Committee of The First Hospital of China Medical University (approval no. 2021-328-2 on June 24 2021), and written informed consent for participation was obtained from all participants.

Cells and culture reagents
HeLa, 293T, TZM-bl, and Jurkat cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (Gibco) or RPMI-1640 medium (Gibco). Both media were supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. Plasmids were transfected into 293T cells using Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer’s instructions. PBMCs obtained from healthy blood donors were purified using Ficoll-Hypaque gradient centrifugation. CD4+ T cells or monocytes were isolated from PBMCs via negative selection using human CD4+ T cells or a CD14+ enrichment cocktail (StemCell Technologies). To stimulate CD4+ T cells, CD3/CD28 activator magnetic beads (Invitrogen) were added to the culture medium along with IL-2 (50 U/mL) (Biomol) for 2 days, in accordance with the manufacturer’s instructions. The isolation and culture of monocytes, monocyte-derived macrophages (MDMs), and monocyte-derived dendritic cells (MDDCs) were performed 40, 41: MDMs were produced by stimulating monocytes with 10 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D) and 50 ng/mL recombinant human macrophage colony-stimulating factor (M-CSF, R&D) for 7 days. MDDCs were generated by incubating CD14-purified monocytes in Iscove’s modified Dulbecco’s medium (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 10 mM HEPES, 1% nonessential amino acids, 1 mM sodium pyruvate, 10 ng/mL GM-CSF, and 50 ng/mL IL-4 (Miltenyi Biotec). On Day 4, two-thirds of the culture medium was replaced with fresh medium containing GM-CSF and IL-4. Immature MDDCs were harvested on Day 6 and used for experiments.

Reagents
IFN-α (Cat# 1103-IF-0101), IFN-β1a (Cat# 8499-IF-010), IFN-γ (Cat# 285-IF-100), and IL-2 (Cat# 202-IL) were purchased from R&D. PHA (Cat# 11249738001) was obtained from Roche.

Plasmids
The ZFP36, ZFP36L1, and ZFP36L2 expression vectors were purchased from OriGene, and their open reading frames were de novo cloned into the pCMV-3Tag-2A (Addgene) vector. Luciferase-expressing Env-defective HIV-1 NL4-3.Luc. R-E-, SIVmac239-luc, HIV-2 Rod-luc, and MLV-luc were provided by Dr. Guangxia Gao 42. HIV-1 proviral NL4-3, BaL, and 89.6 vectors were obtained through the National Institutes of Health AIDS Reagent Program. The HIV-1 proviral vectors of pNL-AD8 were gifted by Dr. Eric Freed. The microRNA-adapted shRNA for ZFP36L2 (Cat#RHS4430-200267269) or control (Cat# RHS4346) was purchased from Dharmacon, the mature antisense shRNA for ZFP36L2 as 5'-TGCACAAGAAGTCGACATC-3'.

RNA interference in Jurkat T cells, CD4+ T cells, and MDMs
Gene silencing of ZFP36L2 was performed using a microRNA-adapted shRNA targeting the open reading frame (Cat# RHS4430-200267269; Dharmacon). A non-targeting shRNA (Cat# RHS4346; Dharmacon) served as the control. Jurkat T cells, pre-activated CD4+ T cells (stimulated with CD3/CD28 and IL-2), and MDMs were transduced with the shRNA-expressing lentivirus 40, 41: at 48 h post-transduction, cells were selected with puromycin for an additional 48 hours. For viral infection, the transduced Jurkat T cells and CD4+ T cells were challenged with replication-competent wild-type HIV-1 NL4-3 or an isogenic Rev (Δ109-116) mutant at a comparable multiplicity of infection (MOI) for 6 hours. Of note, primary CD4+ T cells derived from patients were exempt from this infection step. The cells were then washed twice with pre-cooled phosphate-buffered saline (PBS) to remove residual virus and returned to culture. MDMs were similarly infected with HIV-1 AD8. Culture supernatants were harvested at the indicated time points post-infection. Viral production was quantified by measuring HIV-1 p24 levels using a commercial ELISA kit (ABL) and/or by determining infectious titer using TZM-bl reporter cells, followed by measurement of luciferase activity (Promega). Additionally, the proliferation of transduced Jurkat T cells and CD4+ T cells was assessed using the CellTrace Cell Proliferation Kit (Invitrogen), according to the manufacturer's instructions.

Electroporation of Jurkat T Cells and Primary CD4⁺ T Cells
Jurkat T cells and primary CD4⁺ T cells activated with CD3/CD28 Dynabeads were co-electroporated with either a ZFP36L2 expression plasmid or a mock control plasmid, together with one of the following proviral vectors: NL4-3.Luc.R⁻E⁻, NL4-3, or NL4-3rev Δ(109–116). All transfections were performed using the Lonza 4D-Nucleofector™ System with the 4D-Nucleofector™ X Unit. For Jurkat cells, the SE Cell Line 4D-Nucleofector™ X Kit (Lonza, Cat# V4XC-3024) and program CL-120 were used. For primary CD4⁺ T cells, the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza, Cat# V4XP-3024) was used, with program EO-115 for activated cells 40, 41.

Cytoplasmic and nuclear RNA purification
Cytoplasmic and nuclear RNAs were isolated using a Cytoplasmic & Nuclear RNA Purification Kit (Norgen; Cat# 37400) according to the manufacturer’s protocol. Briefly, cells were lysed using the provided cytoplasmic lysis buffer, followed by centrifugation for the separation of cytoplasmic (supernatant) and nuclear (pellet) fractions. The cytoplasmic RNA was then purified from the supernatant using RNA purification columns, whereas nuclear RNA was extracted from the pellet after disruption with nuclear lysis buffer. Both RNA fractions were treated with DNase I to eliminate genomic DNA contamination. RNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific), and gene expression levels were analyzed by RT-PCR.

Enzyme-linked immunosorbent assay
ELISA was used to measure p24 protein in the culture supernatants (ABL Corporation) according to the manufacturer’s instructions. Test samples were mixed with disruption buffer to inactivate the virus and release HIV-1 p24 into solution for detection. Microtiter wells of a 96-well plate were coated with two murine monoclonal antibodies specific for distinct epitopes on HIV-1 p24. HIV-1 p24 standard solutions or tissue culture test samples were added to the wells, allowing p24 to form immune complexes with the plate-bound antibodies. Unbound materials were then removed by thorough washing. A conjugate solution, containing peroxidase-conjugated human anti-p24 polyclonal antibodies then added to bind the captured HIV-1 p24. After washing the wells to remove the unbound conjugated antibodies, a peroxidase substrate was added. The enzyme–substrate reaction produces a blue color change, which turns yellow upon addition of stop solution. Absorbance was measured at 450 nm. A linear relationship was observed between absorbance and HIV-1 p24 concentration, and sample concentrations were determined by linear regression analysis using the standard curve.

Luciferase detection assay
Luciferase activity was quantified as relative luminescence units (RLUs) in cell lysates using the manufacturer’s instructions (Promega). The TZM-bl indicator cell line was used to quantify HIV-1 infectivity, with luciferase serving as a reporter. Background luminescence from uninfected TZM-bl cells was subtracted from each data point.

Measurement of viral infectivity
HIV-1 p24 levels in culture supernatants were measured using an ELISA (ABL Corporation). To assess viral infectivity, culture supernatants were used to infect TZM-bl reporter cells. Following infection, luciferase activity in cell lysates was measured in RLUs (Promega). Background luminescence from uninfected TZM-bl cells was subtracted from each data point.

HBV nucleocapsid-associated DNA purification
Briefly, secreted HBV particles were purified from culture supernatants using PEG8000 (30% in NaCl) precipitation. The purified relaxed circular DNA from the nucleocapsid-associated DNA was obtained after the following treatments: DNase I digestion to eliminate transfected plasmids, proteinase K and SDS digestion, phenol–chloroform extraction, and isopropanol precipitation. HBV DNA was quantified using qPCR analysis using the primer set 5′-GAATTGATGACTCTAGCTACCTG-3ʹ and 5′-GAAACCACAATAGTTGCCTGATC-3′ 43 .

Southern Blotting
The primer sequences for various spliced forms of HIV mRNA and the probes used were previously described 44. Briefly, cDNAs corresponding to unspliced and singly spliced HIV-1 mRNAs were amplified using primers FP and RP1, while cDNAs for multiply spliced HIV-1 mRNAs were amplified using primers FP and RP2. PCR products were separated on a 1.2% agarose gel, followed by Southern blotting. PCR products amplified from NL4-3.Luc.R-.E- using primers HIV6078F and HIV6621R were used as templates to generate probes for detecting RT-PCR products derived from unspliced and singly spliced mRNAs. Similarly, PCR products amplified using primers HIV8396F and HIV8776R served as templates for probes targeting multiply spliced mRNA RT-PCR products. Probes were labeled with biotin-14-dCTP by random priming (DNA Labeling System, Thermo). The quantity of PCR products was analyzed using the Chemiluminescent Nucleic Acid Detection Module (Thermo).

Primer sequences:
FP: 5′-AGTAAAGCCAGAGGAGATCTCTCG-3′
RP1: 5′-ATTGGTATTAGTATCATTCTTCAAATC-3′
RP2: 5′-GCCACCCATCTTATAGCAAAATCC-3′
HIV6078F: 5′-GTAGCAATAGTAGCATTAGTAGTAGC-3′
HIV6621R: 5′-ATTGGTATTAGTATCATTTCTTCAAATC-3′
HIV8396F: 5′-ACCCGACAGGCCCGAAGGAATAG-3′
HIV8776R: 5′-GCCACCCATCTTATAGCAAAATCC-3′

Co-immunoprecipitation
293T or HeLa cells (5.0 × 106) were lysed using IP lysis buffer (50 mM Tris–HCl, pH 7.2, 50 mM NaCl, 1% NP-40, 1 mM EDTA, 2% glycerol, and 1× protease inhibitor cocktail). The lysates were incubated on ice for 30 min and then centrifuged at 12,000 rpm and 4°C for 10 min. The supernatants were then transferred to fresh tubes, and the pellets were mixed with cold IP lysis buffer prior to sonication, followed by a second round of centrifugation at 12,000 ×g and 4°C for 10 min. The supernatants obtained from the two extraction steps were pooled and incubated with mixed protein A and G Dynabeads (Invitrogen) pretreated overnight with antibodies at 4°C. The IP products were washed with cold IP and PBST buffers 5–10 times (500 µL per wash).

Cytotoxicity assays
The culture supernatants of lentiviral-transduced or untransduced MDMs were collected to assess LDH release. Cytotoxicity was quantified colorimetrically using the Pierce LDH Cytotoxicity Kit (Thermo Fisher Scientific), with LDH activity measured by subtracting A680 nm from A490 nm. Lysis control wells were treated with 10× lysis buffer for 1 h. The cytotoxicity percentage was calculated using a previously described formula 45:
LDH = (LDH lenti-MDM − LDH MDM )/ (LDH Maximum − LDH MDM )  100%
where LDH lenti-MDM or LDH MDM is the amount of LDH activity in the supernatants of lentiviral-treated and untreated cells, respectively, and LDH Maximum represents the activity in the supernatant of the lysis control. LDH activity present in cell-free culture medium was subtracted from all values before normalization.

Expression and purification of recombinant proteins
All recombinant GST-proteins were expressed in BL21(DE3) cells at 37°C and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside for 3.5 h, as described previously46: Cell pellets were lysed by ultrasonication in lysis buffer, clarified by centrifugation at 12,000 ×g for 30 min, and purified by GST-tagged Purification Resin (GST-tag Protein Purification Kit, Beyotime Biotechnology, Beijing, China). All His-ZFP36L2 recombinant proteins were expressed in Rosetta 2 cells and purified on a HisTrap HP column using a 250 mM imidazole gradient (His-tag Protein Purification Kit, Beyotime Biotechnology). All recombinant proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed using western blotting with specific antibodies [mouse monoclonal anti-His (Cell Signaling) and anti-GST (Abcam)].

Subcellular protein fractionation
Nuclear and cytoplasmic protein extracts were isolated using Cell Lysis Buffer (Cell Signaling Technology; Cat# 9803), following the manufacturer’s protocol with minor modifications. Briefly, harvested cells were washed with ice-cold PBS and lysed in 1× Cell Lysis Buffer supplemented with protease and phosphatase inhibitor (Thermo Fisher Scientific; Cat# 78442) for 5 min on ice. Lysates were centrifuged at 14,000 ×g and 4°C for 10 min, and the supernatant (cytoplasmic fraction) was collected. The remaining pellet (nuclear fraction) was resuspended in 1× Cell Lysis Buffer, followed by sonication (30 sec, 20% amplitude, on ice) to disrupt nuclear membranes. After centrifugation (14,000 ×g, 10 min, 4°C), the supernatant (nuclear protein extract) was collected. Protein concentrations were quantified using a BCA assay (Pierce™; Cat# 23225). Protein expression was assessed by western blotting, and GAPDH or Lamin B expression was detected separately as cytoplasmic or nuclear positive controls.

Western blotting and immunoblotting
Western blotting was performed using a standard method to detect cellular proteins. The following antibodies were used: mouse monoclonal anti-ZFP36L2 (1:500, Santa Cruz, Cat# sc-365908), rabbit anti-Lamin B (1:2500, Abcam, Cat# ab16078), mouse anti-nef (1:500, Abcam, Cat# ab42355), Mouse anti-GAPDH (1:1000, ZSGB-BIO, Cat# TA-08), mouse monoclonal anti-FLAG (1:1000, SIGMA, Cat# F1804), mouse anti-His (1:1000, ZSGB-BIO, Cat# TA-02), mouse anti-GST (1:1000, Abcam, Cat# ab92), mouse anti-GFP (1:2500, Abmart, Cat# M20004), rabbit polyclonal anti-p24 (1:1000, Abcam, Cat# ab63913), mouse IgG-HRP (1:5000, Abcam, Cat# ab6789), rabbit IgG-HRP (1:5000, Abcam, Cat# ab6721), TrueBlot® ULTRA Anti-Mouse IgG HRP (1:1000, Rockland, Cat# 18-8817-33), and mouse IgG isotype control (1:1000, Abcam, Cat# 188776).

qPCR
Total RNA was extracted from cells using TRIzol reagent (Invitrogen), in accordance with the manufacturer’s instructions. The obtained RNA was dissolved in 100 µL of DPEC-H2O, and 1 µg of the purified RNA was treated with DNase I (amplification grade, Invitrogen) for 10–15 min at room temperature to remove residual genomic DNA. RNA was immediately primed with oligo-dT and reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen). Real-time PCR was performed using the CT method, with gene expression levels normalized to GAPDH as the internal control. The primers used in this study are listed in Supplementary Table 6.

Statistical analysis
Statistical analyses were performed using GraphPad Prism software (version 8.0.1). Continuous data are presented as the mean ± standard deviation (SD), unless otherwise indicated. Comparisons between two independent groups were performed using an unpaired, two-tailed Student’s t-test. Comparisons among three or more independent groups under a single experimental factor were conducted using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple comparisons, unless otherwise specified. For analyses involving two independent variables, two-way ANOVA was used to assess main effects and interactions. When appropriate, pairwise comparisons were performed using Šidák’s multiple-comparisons test to control the family-wise error rate. A p value < 0.05 was considered statistically significant for all analyses.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Pang et al identifies the RNA-binding protein ZFP36L2 as an IFNb-induced gene that restricts HIV-1 production by blocking the cytoplasmic accumulation of unspliced and singly spliced viral mRNAs. The authors further show that ZFP36L2 interacts with several lentiviral Rev proteins (except FIV) and map determinants in it and HIV-1 Rev. They further show that knockdown of ZFP36L2 enhances HIV replication in macrophages and reverses IFNb-mediated inhibition of viral production in T cells from PLWH.\nThis is a very comprehensive and well performed study and makes a strong case for ZFP36L2 being an antiviral inhibitor of Rev. However, I wonder if the effects of IFNb-induction of ZFP36L2 can be fully explained by Rev inhibition at physiological expression levels. Below are some comments for the authors’ consideration to strengthen the manuscript.\n1. As an RNA-binding, ZFP36L2 will regulate numerous cellular mRNAs. Most of the experiments rely on overexpression of ZFP36L2, which clearly is affecting unspliced/singly spliced mRNAs. However, under endogenous expression conditions (i.e. macrophages) of IFNb induction in T cells is there sufficient ZFP36L2 expressed to yield a measurable change in cytoplasmic mRNA abundance and Gag expression.\n2. Allied to the above, when knocking down ZFP36L2 in macrophages and T cells, what accompanying changes in cellular gene expression is seen. Given that Fig5 suggests the entirety of the inhibition of viral production by IFNb appears to depend on ZPF36L2, it would be good to know if this is really all direct or could be secondary through other known ISGs other than Mx2.\n3. The authors map determinants in HIV-1 Rev and huZFP36L2 for interaction, but what has been mapped in Rev cannot explain the HIV-1/SIV/EIAV Rev interactions but not FIV Rev, nor the potential for species specific interactions. Can any alphafold modelling provide any insight. Can grafting the C-terminus of HIV-1 Rev onto FIV provide a gain of restriction?\nMinor:\n1. The correlation between ZFP36L2 and viral load is unlikely to be specific. You would find that with any ISG/IFN-dependent process.\n2. There is a lot of important data in the supplement that would be better in the main figures.", "reviewer_2": "The study identifies ZFP36L2 as inteferon-ß induced factor that binds to HIV-1 Rev, inhibits the nuclear export of unspliced HIV-1 mRNAs that contain the RRE and thereby blocks HIV-1 replication. This study is highly interesting as it identifies to my knowledge a unique Rev-dependent HIV-1 restriction factor that is on top specific for interferon-ß but not other interferons.\nThe data is very solid and supports the conclusions by employing ZFP36L2 overexpression and depletion in primary CD4+ T cells as well macrophages. They further even use these techniques in CD4+ T cells from PLWH that are on ART or not.\nFurthermore, by comprehensive analysis of several Rev variants from different lentiviruses they manage to identify the ZFP36L2 binding regions in Rev and finally generate a Rev variant that is resistant towards inhibition by ZFP36L2 but still retains the ability to replicate.\nThere are a few aspects that might help to enhance the clarity and further support the overall convincing data.\nEx.Fig.2a:\nIt would by highly interesting to see the expression levels of ZFP36L2 in primary resting CD4+ T cells and to analyze if the levels go down upon T cell stimulation. If the levels are high in resting CD4+ T cells, would depletion of ZFP36L2 render resting T cells permissive towards HIV-1 infection?\nFig.2a, lines 175-178: why is no effect on luc activity an indicator for late effects? For luc activity, the virus needs RT, integration, gene expression; so I feel this is not a good argument to discard early effects\nData Rev-Interaction with ZFP36L2, can you model the interaction with alpha-fold? Does it mirror your in vitro results? Can you narrow down the specific aa that lead to interaction with ZFP36L2? Can you generate a gain of function for Revs that do not interact with ZFP36L2?\nHow is the subcellular localization of ZFP36L2 and Rev?\nWhat really striked me is the fact ZFP36L2 knockout by shRNA nearly completely reverted the inhibitory effect by IFN-ß (shown in several figures in primary CD4+ T cells, macrophages and patient samples). Do the authors hence imply that IFNb induced antiviral acitivty towards HIV-1 is nearly solely attributed to induction of ZFP36L2? What about all the other known ISGs and interferon-induced restriction factors that suppress HIV-1?\nPlease explain where the ART naive HIV-1 patient samples come from. As far as I am aware of, nowadays it is state-of-the art to treat patients as soon as they are diagnosed with HIV-1\nFig5; I would have expected a much higher differene in viral titers when CD4+ T cells from ART-treated vs ART-naive HIV-1 patients are analyzed\nWhat impressed me in this study is that they are apparently able to knock-down cellular factors in primary CD4+T cells and macrophages by shRNA-lentiviral transduction. This is extremely challenging, especially when it is done in patient material. Unfortunately, the Material&Methods section falls completely short to explain this methodology. It is not even mentioned there. Same thing with electroporation of expression plasmids in primary cells. Please describe precisely, so that the experiments can be repeated and validated. Give the shRNA targeting sequences.\nI do not think that it is correct to use students t test when comparing more than two groups. Then ANOVA with post tests should be employed. Some experiments only show data from one biological replicate with triplicates. Standard is three biological replicates.\nDo you find any variations in Rev, in natural HIV-1 sequences from patients (see multiple databases, ie LosAlamos) that might indicate adaptation towards ZFP36L2 resistance in vivo? Are there Rev polymorphisms that are associated with different courses of HIV-1 infection/AIDS progression you could associate with ZFP36L2 resistance?\nLikewise, are there known polymorphisms SNPs in ZFP36L2 that are associated with disease?\nI think the discussion is very close to the results, mainly reiterating them.\nMinor:\n-Lines 98-199 remove statement; seems odd in the Lenacapavir era\n-lines 108, the data is shown in fig 1a and b I think\n-lines 184-189, omit this explanation, not needed\n-ExFig.7 is not mentioned/introduced in the main text", "reviewer_3": "The manuscript “ZFP36L2, an interferon beta-induced inhibitor, restricts the nuclear export of HIV-1 transcripts” by Pang et al. presents evidence for ZFP36L contributing to the antiviral, IFN-beta-mediated response to HIV-1 infection. Other viruses inhibited by this factor include HIV-2, SIV, EIAV, but not FIV and MLV. This is shown by knockout in the context of IFN-beta treatment, and heterologous expression experiments in cell lines and in CD4+ T-cells from PLHIV. In vivo ZFP36L2 mRNA levels inversely correlated with viremia. ZFP36L2 binds to Rev and interferes with nuclear export of unspliced and partially spliced HIV-1 mRNAs. They mapped the Rev-binding site within ZFB36L2 and vice versa. Together, I believe this is an exciting and carefully executed study.\nMajor concerns: I don’ t have major concerns and I commend the authors to this beautiful and extensive study.\nMinor concerns:\n-not an essential requirement, but the reader wonders what made the authors start studying ZFP36L2. Some sort of explanation would benefit the start of the flow of the paper.\n-the discussion is in wide parts a reiteration of the results. Instead of repeating results, results should be contextualised.\n-Extended Fig. 1a) “At 48 h post-transfection, HIV-1 capsids in culture were quantified using p24 ELISA”. I don’ t think capsids are quantified, it’ s rather the mass of capsid protein.\n-Extended Fig. 1 c-e, “Jurkat cells were infected with 100 ng of HIV-1NL4-3”. I believe cells weren’ t infected with 100 ng of HIV-1, rather 100 ng of p24 equivalents.\n-I acknowledge the nuclear/cytoplasmic separation was validated at the protein level (Fig. 2c), however apparently not at the RNA level (Fig. 2b). Showing purity of the nuclear and cytoplasmic compartment by amplifying a strictly nuclear and almost exclusively cytoplasmically located RNAs would be beneficial.\n-Immunoblots shown in Fig. 3 and extended Fig. 3 seem to imply that SFP36L2 is located both in the cytoplasm and nucleus. Does it have an NLS?\n-the finding of the delta109–116 Rev mutant evading ZFP36L2 inhibition suggests HV-1 could evolve to evade restriction while maintaining a functional Rev – can this be recapitulated in virus passaging experiments in ZFP36L2-overexpressing cells? Looking at different subtypes, is the 109-116 Rev region polymorphic?\nLines 403-404: “ZFP36L2 transcript levels were inversely correlated with plasma HIV-1 RNA levels (Fig. 5b), supporting its role as a negative regulator of viral replication.” Although a causal link is a tempting hypothesis, I think it is more likely (or also likely) that ZFP36L2 expression is merely a surrogate of ISG expression in general during unsuppressed HIV-1 infection. In other words, one would expect to see increased expression of any ISG, antiviral or not, in untreated PLHIV. I suggest to rephrase more carefully.\n-the experiments shown in Fig. 5 g (reconstitution of ZFP36L2 in primary cells) don’ t exclude the possibility that the factor was expressed at aberrantly high levels and that this high overexpression is causing some antiviral state per se. It would have been nice to include some specificity control, like expressing the ZFP36L2 mutant that is no longer able to bind to Rev, as a negative control (instead of GFP)\nThe methods seem to lack a chapter about the lentiviral transduction of different cell types, especially primary T-cells and macrophages."}, "author_response": "Responses to Reviewers’ Comments\nWe thank the reviewers for their comments and suggestions, which have helped us\nsignificantly improve our manuscript. We highlighted all changes in red in the revised\nmanuscript and provided our point-by-point responses to the reviewers’ comments\nbelow.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Pang et al identifies the RNA-binding protein ZFP36L2 as an IFNb-\ninduced gene that restricts HIV-1 production by blocking the cytoplasmic accumulation\nof unspliced and singly spliced viral mRNAs. The authors further show that ZFP36L2\ninteracts with several lentiviral Rev proteins (except FIV) and map determinants in it and\nHIV-1 Rev. They further show that knockdown of ZFP36L2 enhances HIV replication in\nmacrophages and reverses IFNb-mediated inhibition of viral production in T cells from\nPLWH.\nThis is a very comprehensive and well performed study and makes a strong case for\nZFP36L2 being an antiviral inhibitor of Rev. However, I wonder if the effects of IFNb-\ninduction of ZFP36L2 can be fully explained by Rev inhibition at physiological\nexpression levels. Below are some comments for the authors’ consideration to\nstrengthen the manuscript.\n1. As an RNA-binding, ZFP36L2 will regulate numerous cellular mRNAs. Most of the\nexperiments rely on overexpression of ZFP36L2, which clearly is affecting\nunspliced/singly spliced mRNAs. However, under endogenous expression conditions\n(i.e. macrophages) of IFNb induction in T cells is there sufficient ZFP36L2 expressed to\nyield a measurable change in cytoplasmic mRNA abundance and Gag expression.\nResponse 1: We appreciate your thoughtful suggestion.\nIn this revision, we followed your suggestion and examined whether endogenous\nZFP36L2 inhibits Gag nuclear export in primary macrophages. As shown in the new\nSupplementary Fig. 8c–e, depletion of endogenous ZFP36L2 promotes Gag—but not\nRev—RNA nuclear export in primary macrophages. Conversely, when endogenous\nZFP36L2 is present, Gag RNA export from the nucleus to the cytoplasm is inhibited,\nwhile Rev RNA export remains unaffected. Therefore, endogenous ZFP36L2 in\nmacrophages also suppresses Gag RNA nuclear export. This has now been described\nin the main text (Page 15, lines 326–328).\n2. Allied to the above, when knocking down ZFP36L2 in macrophages and T cells, what\naccompanying changes in cellular gene expression is seen. Given that Fig5 suggests\nthe entirety of the inhibition of viral production by IFNb appears to depend on ZPF36L2,\nit would be good to know if this is really all direct or could be secondary through other\nknown ISGs other than Mx2.\nResponse 2: Thank you for this question—we are happy to address it.\nWe did not intend to suggest that IFN-β relies entirely on ZFP36L2 to inhibit HIV-1\ninfection in either CD4⁺ T cells or primary macrophages. It is well established that IFN-β\ninduces MX2, a host restriction factor; therefore, at least both MX2 and ZFP36L2\nfunction as IFN-β–mediated antiviral effectors against HIV-1 infection. To specifically\nevaluate the contribution of ZFP36L2, we took advantage of the distinct expression\nkinetics of these two genes: ZFP36L2 expression peaks at 48 h post-treatment,\nwhereas MX2 levels decline. Using this approach, we assessed IFN-β antiviral activity\nunder ZFP36L2-depleted conditions. As shown in Fig. 5a,b and Supplementary Fig.\n8c,d in this revision, depletion of ZFP36L2 did not completely abrogate the inhibitory\neffect of IFN-β in either CD4⁺ T cells or primary macrophages.\nFurthermore, we transfected proviral HIV-1 vectors into these host cells. In this\nsystem, HIV-1 proceeds only through the late stages of replication, during which MX2\nhas no effect, as it acts early in the viral life cycle. This allowed us to specifically assess\nthe role of endogenous ZFP36L2 in the presence of IFN-β. As shown in Supplementary\nFig. 9a-d in this revision, ZFP36L2 depletion again did not fully eliminate IFN-β-\nmediated inhibition, indicating that IFN-β does not depend exclusively on ZFP36L2 to\nsuppress HIV-1 replication during the late phase. Importantly, IFN-β also inhibited\ninfection of RevΔ(109–116) mutant HIV-1, which is resistant to ZFP36L2, as shown in\nFig. 5c,d and Supplementary Fig. 9c, further demonstrating that IFN-β does not rely\nsolely on ZFP36L2. Therefore, we did not intend to convey that IFN-β completely\ndepends on ZFP36L2 for antiviral activity.\nRegarding Fig. 5i,l in the previous submission (now Fig. 6i,l in the revised\nmanuscript), ZFP36L2 depletion did not fully abrogate IFN-β–mediated suppression.\nThese differences may not have been readily apparent because numerical values were\npresented using exponents. Notably, in the absence of ZFP36L2, IFN-β-mediated\ninhibition was reduced from 3.75-fold to 1.4-fold in ART-naïve patients, and from 4.7-\nfold to 1.28-fold in ART-treated patients. Moreover, as shown in Fig. 6e, primary CD4⁺ T\ncells from patients were transduced with lentiviral shRNA. Under these experimental\nconditions, most HIV-1 genomes are expected to be integrated proviruses, similar to\nHIV-1 producer cells; therefore, MX2—an early-acting restriction factor—may have\nlimited activity. Overall, we did not propose that IFN-β relies exclusively on ZFP36L2 to\ninhibit HIV-1 infection, and we have now clarified this point in the Discussion section in\nthis revised manuscript (Page 21, Lines 467–469).\n3. The authors map determinants in HIV-1 Rev and huZFP36L2 for interaction, but what\nhas been mapped in Rev cannot explain the HIV-1/SIV/EIAV Rev interactions but not\nFIV Rev, nor the potential for species specific interactions. Can any alphafold modelling\nprovide any insight. Can grafting the C-terminus of HIV-1 Rev onto FIV provide a gain of\nrestriction?\nResponse 3: We thank the reviewer for the insightful suggestion regarding the use of\nAlphaFold modeling to investigate species-specific determinants of the Rev–ZFP36L2\ninteraction. We are happy to address this point.\n(1) We used AlphaFold3 to predict structural models of ZFP36L2 in complex with\nHIV-1, SIV, EIAV, and FIV Rev proteins. However, all predicted complexes yielded low\ninterface predicted template modeling (ipTM) scores, indicating low confidence in the\npredicted protein–protein interaction interfaces. This limitation likely reflects intrinsic\nfeatures of Rev—its dependence on oligomerization and the high conformational\nflexibility of its C-terminal intrinsically disordered region—features that current structure-\nprediction algorithms do not robustly capture (PMID: 38718835; PMID: 39824781). As a\nresult, the models did not provide reliable structural insight into the observed specificity\nof interaction, including why ZFP36L2 binds HIV-1, SIV, and EIAV Rev but not FIV Rev.\nImportantly, our experimentally mapped interaction determinants—defined through\nfunctional assays in both Rev and ZFP36L2—offer a clear and experimentally\nsupported explanation for this species-specific interaction pattern (Supplementary Fig.\n4–6). Thus, while computational modeling can be informative, our experimental data\ncurrently provide the most reliable mechanistic basis for understanding differential\ninteractions among Rev orthologs.\n(2) Following your suggestion, we grafted the C-terminus of HIV-1 Rev (residues 109–\n116) onto FIV Rev and performed co-immunoprecipitation assays to test whether this\nmodification could confer ZFP36L2 binding. As shown in the following figure, the grafted\nFIV Rev did not interact with ZFP36L2. In contrast, wild-type HIV-1 Rev robustly bound\nZFP36L2, whereas wild-type FIV Rev showed no detectable interaction. These results\nindicate that the HIV-1 Rev residues 109–116 alone are not sufficient to confer\nZFP36L2 binding when grafted onto FIV Rev. It is possible that this C-terminal motif\ncontributes to ZFP36L2 interaction only in the context of the full HIV-1 Rev protein,\npotentially by influencing Rev oligomerization or higher-order structural conformation.\nConsequently, simple grafting of this region is insufficient to enable FIV Rev to bind\nZFP36L2. Further studies will be required to determine why FIV Rev fails to interact with\nZFP36L2. More extensive domain swaps or systematic exchange of additional HIV-1\nand FIV Rev sequences may help to identify the structural or sequence determinants\nresponsible for ZFP36L2 recognition.\nMinor:\n1. The correlation between ZFP36L2 and viral load is unlikely to be specific. You would\nfind that with any ISG/IFN-dependent process.\nResponse 4: We agree with your point and have revised the sentence as follows:\n“ZFP36L2 transcript levels were inversely correlated with plasma HIV-1 RNA levels (Fig.\n6b). This correlation may not be specific, as ZFP36L2 is an IFN-stimulated gene, and\nsimilar correlations may also be observed for other IFN-induced genes” in this revision\n(Page 18, Lines 399–402).\n2. There is a lot of important data in the supplement that would be better in the main\nfigures.\nResponse 5: Following your recommendation, we have moved the data previously\npresented in Extended Data Fig. 8a–i to the main figures (now shown as new Fig. 4). In\naddition, Extended Data Fig. 8j,k have been incorporated into the new Supplementary\nFig. 7c,d, and the former Extended Data Figs. 9 and 10 have been combined into a new\nSupplementary Fig. 8 in this revision.\nReviewer #2 (Remarks to the Author):\nThe study identifies ZFP36L2 as inteferon-ß induced factor that binds to HIV-1 Rev,\ninhibits the nuclear export of unspliced HIV-1 mRNAs that contain the RRE and thereby\nblocks HIV-1 replication. This study is highly interesting as it identifies to my knowledge\na unique Rev-dependent HIV-1 restriction factor that is on top specific for interferon-ß\nbut not other interferons.\nThe data is very solid and supports the conclusions by employing ZFP36L2\noverexpression and depletion in primary CD4+ T cells as well macrophages. They\nfurther even use these techniques in CD4+ T cells from PLWH that are on ART or not.\nFurthermore, by comprehensive analysis of several Rev variants from different\nlentiviruses they manage to identify the ZFP36L2 binding regions in Rev and finally\ngenerate a Rev variant that is resistant towards inhibition by ZFP36L2 but still retains\nthe ability to replicate.\nThere are a few aspects that might help to enhance the clarity and further support the\noverall convincing data.\nEx.Fig.2a:\nIt would by highly interesting to see the expression levels of ZFP36L2 in primary resting\nCD4+ T cells and to analyze if the levels go down upon T cell stimulation. If the levels\nare high in resting CD4+ T cells, would depletion of ZFP36L2 render resting T cells\npermissive towards HIV-1 infection?\nResponse 1: Thank you for this question. We followed your suggestion and examined\nZFP36L2 expression in primary resting CD4⁺ T cells. As shown in the accompanying\nfigure (Review Fig. 1), ZFP36L2 is indeed expressed in resting CD4⁺ T cells and is\ndownregulated upon T-cell stimulation. In the absence of ZFP36L2, cytoplasmic Gag\nRNA levels increased while nuclear Gag RNA levels decreased, whereas multiply\nspliced Rev RNA remained unchanged in both cytoplasmic and nuclear fractions. These\nobservations indicate that ZFP36L2 functions to inhibit HIV-1 Gag RNA nuclear export\nin resting CD4+ T cells. Although these findings support the reviewer’s hypothesis, we\nfeel that a full exploration of ZFP36L2 function specifically in primary resting CD4⁺ T\ncells is beyond the scope of the current study and would be more appropriate for a\nsubsequent investigation. We hope that the additional data provided here will\nnonetheless satisfy the reviewer’s request.\nReview Fig. 1. Silencing ZFP36L2 enhances cytoplasmic Gag RNA levels in resting CD4⁺\nT cells.\na, ZFP36L2 is downregulated upon T-cell stimulation. Resting CD4⁺ T cells were stimulated with\nor without CD3/CD28 and IL-2 for the indicated durations, then lysed for Western blot analysis of\nZFP36L2 and GAPDH. b,c, Resting CD4⁺ T cells were electroporated with siRNA targeting\nZFP36L2 or a control siRNA together with the NL4-3-Luc.R⁻E⁻ proviral vector. Three days post-\nelectroporation, cells were lysed and nuclear and cytoplasmic RNA fractions were isolated for\nRT–qPCR quantification of HIV-1 transcripts, normalized to GAPDH, in the presence or absence\nof reverse transcriptase (b). Western blotting was performed to assess ZFP36L2, Lamin B, and\nGAPDH levels in nuclear and cytoplasmic fractions (c). P < 0.01, *P < 0.05 (two-tailed unpaired\nStudent’s t-test); data are shown as mean ± SD from three triplicates.\nFig.2a, lines 175-178: why is no effect on luc activity an indicator for late effects? For luc\nactivity, the virus needs RT, integration, gene expression; so I feel this is not a good\nargument to discard early effects\nResponse 2: Thank you for this point. We have followed you suggestion and deleted\nthese unnecessary words in this revision (Page 8, Line 178).\nData Rev-Interaction with ZFP36L2, can you model the interaction with alpha-fold?\nDoes it mirror your in vitro results? Can you narrow down the specific aa that lead to\ninteraction with ZFP36L2? Can you generate a gain of function for Revs that do not\ninteract with ZFP36L2?\nHow is the subcellular localization of ZFP36L2 and Rev?\nResponse 3: Thank you for this relevant suggestion.\n(1) Following your recommendation, we used AlphaFold to model the Rev–ZFP36L2\ninteraction. However, all predicted Rev–ZFP36L2 complexes produced low interface\npredicted template modeling (ipTM) scores, indicating low confidence in the interaction\ninterface. This limitation likely reflects intrinsic properties of Rev—its requirement for\noligomerization and the pronounced conformational flexibility of its C-terminal\nintrinsically disordered region—features that current structure-prediction algorithms do\nnot reliably model (PMID: 38718835; PMID: 39824781). Consequently, the AlphaFold\npredictions did not yield structural insight into the specificity of interaction, including why\nZFP36L2 binds HIV-1, SIV, and EIAV Rev but not FIV Rev. Please also see our\nResponse 3 to Reviewer 1 for additional discussion.\n(2) Regarding your point about generating a gain-of-function Rev, we have indeed\nproduced mutant Rev proteins in this study. Among these mutants, the Δ109–116 Rev\nvariant—which supports Rev-defective HIV replication—does not bind to ZFP36L2\n(Supplementary Fig. 7a) and confers resistance to ZFP36L2-mediated inhibition both in\nimmortalized cell lines and in primary CD4+ T cells (Fig. 4a–i). Furthermore, as shown\nin Supplementary Fig. 7c,d, the Δ109–116 Rev mutant fails to enable ZFP36L2 binding\nto viral RNA. Collectively, these findings indicate that we have identified a gain-of-\nfunction Rev variant that does not interact with ZFP36L2. Because the 109–116 region\ncomprises only seven amino acids and has already been defined in the present study,\npinpointing the specific residue(s) responsible for this interaction will be best addressed\nin future work. We hope it can be acceptable to the reviewers.\n(3) ZFP36L2 is known to shuttle between the nucleus and the cytoplasm, as previously\nreported (PMID: 34611029; PMID: 16508015; PMID: 7958838). Rev is also a\nnucleocytoplasmic shuttling protein. As shown in Fig. 2c,h; Fig. 3c,e,i; and\nSupplementary Fig. 3c,f, both Rev and ZFP36L2 localize to the nucleus and the\ncytoplasm. Consistently, Fig. 3f and Supplementary Fig. 4b–d and Supplementary Fig.\n5b illustrate that Rev interacts with ZFP36L2 in cells. Collectively, these results\ndemonstrate that ZFP36L2 and Rev are nucleocytoplasmic shuttling proteins and that\nthey can interact with each other.\nWhat really striked me is the fact ZFP36L2 knockout by shRNA nearly completely\nreverted the inhibitory effect by IFN-ß (shown in several figures in primary CD4+ T cells,\nmacrophages and patient samples). Do the authors hence imply that IFNb induced\nantiviral acitivty towards HIV-1 is nearly solely attributed to induction of ZFP36L2?\nWhat about all the other known ISGs and interferon-induced restriction factors that\nsuppress HIV-1?\nResponse 5: Thank you for this question, and we are glad to address it.\nIn this study, we did not intend to suggest that IFN-β relies exclusively on ZFP36L2\nto inhibit HIV infection in either CD4⁺ T cells or primary macrophages. As shown in\nSupplementary Fig. 2h,i, IFN-β also induces MX2, a well-established host restriction\nfactor, in addition to ZFP36L2. In several experiments (those labeled “transfection” or\n“electroporation”), we introduced proviral HIV-1 vectors directly into host cells. Under\nthese conditions, HIV-1 undergoes only the late phase of replication, during which MX2\nhas no antiviral effect because it acts early in the viral life cycle. This allowed us to\nspecifically examine the role of endogenous ZFP36L2 in the presence of IFN-β. In this\ncontext, ZFP36L2 knockdown partially, but not completely, reversed IFN-β–mediated\ninhibition.\nIn the virus infection experiments, to specifically evaluate the contribution of\nZFP36L2, we took advantage of the different expression kinetics of the two genes:\nZFP36L2 expression peaks 48 h after IFN-β treatment, whereas MX2 levels decrease.\nThis strategy minimized MX2’s antiviral activity as much as possible, thereby allowing\nus to focus on ZFP36L2. Under these conditions, ZFP36L2 knockdown again appeared\nto relieve IFN-β–mediated inhibition.\nMoreover, IFN-β also inhibited infection by the RevΔ(109–116) HIV-1 mutant,\nwhich is resistant to ZFP36L2, as shown in Fig. 5c,d and Supplementary Fig. 9c.\nThis\nfurther demonstrates that IFN-β does not act solely through ZFP36L2. Therefore, we did\nnot intend to convey that IFN-β depends entirely on ZFP36L2 for its antiviral activity.\nPlease also refer to Response 2 to Reviewer 1.\nPlease explain where the ART naive HIV-1 patient samples come from. As far as I am\naware of, nowadays it is state-of-the art to treat patients as soon as they are diagnosed\nwith HIV-1\nResponse 6: Thank you for this comment. When individuals first arrive at the hospital,\nwe collect blood samples for diagnostic testing to determine whether they are infected\nwith HIV. Before the diagnosis is confirmed, we isolate and cryopreserve their PBMCs.\nIf the test result later turns out to be positive, these stored PBMCs are then classified as\nsamples from ART-naïve HIV-1–infected individuals.\nFig5; I would have expected a much higher differene in viral titers when CD4+ T cells\nfrom ART-treated vs ART-naive HIV-1 patients are analyzed\nResponse 7: Thank you for this question, and we are glad to address it.\nFirst, as shown in Fig. 6g, 6i, and 6l (corresponding to Fig. 5 in the previous\nmanuscript), virion production from ART-naive HIV-1–infected individuals was indeed\nhigher than that from ART-treated individuals (approximately 15,000–50,000 RLU\nversus ~4,000 RLU). The reason why this difference is not more pronounced likely\nrelates to several factors:\n(1) heterogeneity of viral populations.\nIn both ART-treated and ART-naive HIV-positive individuals, the viral populations are\nheterogeneous. Some patients have high viral loads (Supplementary Table 3), some\nharbor defective viruses that cannot replicate, and others carry viruses under strong\nantiretroviral pressure. Because the patients in this study were randomly selected rather\nthan matched as a cohort, direct quantitative comparisons between groups naturally\nshow considerable variability.\n(2) differences in CD4⁺ T-cell quality and abundance.\nCD4⁺ T cells from ART-naive patients often differ substantially from those of ART-\ntreated individuals. ART-naive patients usually have much lower CD4⁺ T-cell counts and\npoorer cellular health. These cells were stimulated with CD3/CD28 activators before\nlentiviral transduction (Fig. 6e), and these underlying differences likely influenced ex\nvivo viral replication. Therefore, a uniformly large increase in virus production from ART-\nnaive samples was not necessarily expected.\n(3) use of TZM-bl reporter cells for detection.\nVirion production was quantified using TZM-bl cells, which are more sensitive than p24\nELISA for detecting low levels of infectious HIV-1. Importantly, TZM-bl cells\npreferentially detect replication-competent virus. Defective virions produced by CD4⁺ T\ncells are not measured by this assay, which may reduce the apparent difference\nbetween ART-naive and ART-treated groups.\nIn summary, although CD4⁺ T cells from ART-naive individuals generally produced\nhigher levels of virus than those from ART-treated patients, biological variability,\ndifferences in CD4⁺ T-cell condition, and the detection method used all contribute to the\nmodest magnitude of the observed difference in our ex vivo assay system.\nWhat impressed me in this study is that they are apparently able to knock-down cellular\nfactors in primary CD4+T cells and macrophages by shRNA-lentiviral transduction. This\nis extremely challenging, especially when it is done in patient material. Unfortunately,\nthe Material&Methods section falls completely short to explain this methodology. It is not\neven mentioned there. Same thing with electroporation of expression plasmids in\nprimary cells. Please describe precisely, so that the experiments can be repeated and\nvalidated. Give the shRNA targeting sequences.\nResponse 8: Thank you for bringing this to our attention. Following your suggestion, we\nhave added all the requested information in the revised manuscript. The procedures for\nshRNA knockdown and plasmid electroporation in primary cells, including CD4⁺ T cells\nand macrophages, are well-established in our laboratory. We have cited our previously\npublished papers that describe these methods in detail (Refs. 40, 41, 46) and have now\nincluded descriptions in the Methods section (Pages 30–32, Lines 7045–732).\nAdditionally, we have provided the shRNA targeting sequences in this revision (Page\n30, Line 700).\nI do not think that it is correct to use students t test when comparing more than two\ngroups. Then ANOVA with post tests should be employed. Some experiments only\nshow data from one biological replicate with triplicates. Standard is three biological\nreplicates.\nResponse 9: Thank you for raising this point. Following your suggestion, we have\nadjusted the description about statistical method in the revised manuscript as follow:\n“Statistical analyses were performed using GraphPad Prism software (version 8.0.1).\nContinuous data are presented as the mean ± standard deviation (SD), unless\notherwise indicated. Comparisons between two independent groups were performed\nusing an unpaired, two-tailed Student’s t-test. Comparisons among three or more\nindependent groups under a single experimental factor were conducted using one-way\nanalysis of variance (ANOVA), followed by Tukey’s post hoc test for multiple\ncomparisons, unless otherwise specified. For analyses involving two independent\nvariables, two-way ANOVA was used to assess main effects and interactions. When\nappropriate, pairwise comparisons were performed using Šidák’s multiple-comparisons\ntest to control the family-wise error rate. A P value < 0.05 was considered statistically\nsignificant for all analyses.” (Page 38, Lines 876–886)\nIn addition, we have revised the figure legends at the relevant positions (marked in\nred in the revised manuscript) to clarify the statistical tests used and the number of\nreplicates. Although some datasets may appear to reflect a single biological replicate\ndue to low variability and correspondingly small standard deviation values, all\nquantitative results shown are derived from at least three independent biological\nreplicates. This information can be verified in the Source Data files provided with the\nrevised manuscript.\nDo you find any variations in Rev, in natural HIV-1 sequences from patients (see\nmultiple databases, ie LosAlamos) that might indicate adaptation towards ZFP36L2\nresistance in vivo? Are there Rev polymorphisms that are associated with different\ncourses of HIV-1 infection/AIDS progression you could associate with ZFP36L2\nresistance?\nLikewise, are there known polymorphisms SNPs in ZFP36L2 that are associated with\ndisease?\nResponse 10: Thank you for this interesting question; we are glad to address it.\n(1) To investigate whether natural Rev variation suggests in vivo adaptation toward\nZFP36L2 resistance, we analyzed patient-derived HIV-1 Rev sequences from the Los\nAlamos HIV Sequence Database. We used the 2022 curated group M Rev protein\nalignment, which contains 5,901 sequences. Focusing on the 109–116 region examined\nin this study, 5,305 of 5,901 sequences (89.9%) encode the full eight-residue motif, 149\nsequences (2.5%) show partial deletions or premature stop codons within this region,\nand 447 sequences (7.6%) lack the entire segment (see Review Table 1). The\nconsensus sequence across isolates is VLGSGTKE. Residues 110, 113, and 115 are\nhighly conserved (L, G, and K at 99.7%, 92.9%, and 86.1%, respectively), whereas\npositions 109, 111, 112, 114, and 116 show modest variability. A sequence-logo\nrepresentation of residues 109–116 is provided in Review Figure 2.\nThe consensus sequence across patient isolates is VLGSGTKE. Residues 110, 113,\nand 115 are highly conserved (L, G, and K at 99.7%, 92.9%, and 86.1%, respectively),\nwhile positions 109, 111, 112, 114, and 116 display modest variability. A sequence-logo\nrepresentation of residues 109–116 is shown in the following Review Figure 2.\n(2) Regarding associations between Rev polymorphisms and clinical outcome: we\nretrieved all available long-term non-progressor (LTNP; n = 12,925) and rapid\nprogressor (RP; n = 5,353) sequences from the LANL HIV Sequence Database. After\ndeduplication by patient identifier (retaining the earliest submission per patient), 801\nunique LTNP and 391 unique RP patient entries were obtained. Rev sequences were\nextracted using GeneCutter, and entries lacking Rev coverage or containing only gaps\nacross the Rev region were excluded, yielding 122 LTNP and 54 RP sequences.\nBecause the original experimental designs and raw chromatogram data were\nunavailable, we could not reliably distinguish true biological deletions of the 8–amino\nacid Rev motif VLESGTKE (HXB2 positions 109–116) from artefacts resulting from\namplicon design or incomplete sequencing coverage. To avoid overinterpretation of\nthese ambiguous cases, we restricted motif-based analyses to sequences with intact\nalignment across this region and excluded obviously abnormal sequences as well as\nthose entirely lacking the motif. This stringent curation resulted in a final dataset of 43\nLTNP and 28 RP sequences with reliable coverage of the VLESGTKE motif.\nWithin this curated dataset, we generated separate sequence logos for LTNP and\nRP sequences and quantified the amino acid distribution at each of the eight motif\npositions in both groups (see Review Figure 3). To summarize motif-level patterns, each\nsequence was classified as wild type (perfectly matching VLESGTKE), single-\nsubstitution variant (exactly one amino acid substitution within the motif), or multiple-\nsubstitution variant (two or more substitutions within the motif). Among the 71\nsequences analyzed (43 LTNP and 28 RP), Rev motif variants were more frequent in\nRP than in LTNP (see Review Table 2). Any Rev motif variant (single or multiple\nsubstitution) was detected in 89.3% (25/28) of RP sequences compared with 53.5%\n(23/43) of LTNP sequences (Fisher’s exact test, p = 0.0018). Multiple-substitution\nvariants were particularly enriched in RP, observed in 64.3% (18/28) of RP sequences\nversus 30.2% (13/43) of LTNP sequences (p = 0.0070), whereas the frequency of\nsingle-substitution variants was similar between the two groups (25.0% vs. 23.3% in RP\nand LTNP, respectively). Conversely, wild-type Rev motifs were substantially more\ncommon in LTNP than in RP (46.5% vs. 10.7%).\nThese findings suggest that variation within the Rev 109–116 motif may be\nassociated with disease progression. However, this analysis is preliminary. Determining\nwhether Rev evolves in vivo to escape ZFP36L2—and whether such polymorphisms\nare linked to distinct clinical courses of HIV infection—is beyond the scope of the\npresent study and will require future investigations, such as deep viral sequencing\ncombined with detailed clinical phenotyping in well-characterized patient cohorts.\n(3) Regarding associations between ZFP36L2 polymorphisms and HIV disease\nprogression: Our current dataset and the scope of this study do not include clinical\nmetadata (e.g., viral load, CD4⁺ T-cell decline rate, GWAS data, or progression to AIDS)\nfor the sequenced isolates. As a result, we cannot assess whether naturally occurring\nZFP36L2 variants are associated with differential HIV susceptibility or clinical outcomes.\nAddressing this question will require population-scale genetic analyses linked to\nlongitudinal clinical data.\nTherefore, evaluating associations between Rev or ZFP36L2 polymorphisms and\nHIV clinical outcomes falls beyond the scope of the present study but represents an\nimportant direction for future work. We have now added this point to the Discussion\nsection in the revised manuscript (Pages 23–24, Lines 524–529). We hope this\nclarification is acceptable to the reviewers.\nI think the discussion is very close to the results, mainly reiterating them.\nResponse 11: Thank you for this suggestion. We have revised the Discussion section\nto reduce repetition of the Results and to better contextualize our findings. Specifically,\nwe have incorporated additional hypotheses and interpretations to strengthen the\ndiscussion in the revised manuscript (Page 21, Lines 467–469; Page 22, Lines 487–\n489; Pages 23-24, Lines 524–529; Page 24, Lines 536–538).\nMinor:\n-Lines 98-199 remove statement; seems odd in the Lenacapavir era\nResponse 12: Thank you for this recommendation, and we have removed the\nstatement as suggested. The revised sentence now reads:\n“Therefore, ZFP36L2 is a critical effector of the anti–HIV-1 activity mediated by type I\nIFN-β, shedding new light on the intricate molecular mechanisms underlying the IFN-β–\ndriven antiviral response against HIV-1” in this revision (Page 5, Lines 98-100).\n-lines 108, the data is shown in fig 1a and b I think\nResponse 13: In the previously submitted manuscript, line 108 referred to our\nexamination of the ZFP36 family members in relation to HIV-1, which was correct.\nAccordingly, the relevant data are now presented in Supplementary Fig. 1a,b in the\nrevised version of the manuscript (Page 5, Line 108).\n-lines 184-189, omit this explanation, not needed\nResponse 13: We have removed the redundant explanation in lines 184–189 in the\nrevised manuscript.\n-ExFig.7 is not mentioned/introduced in the main text\nResponse 14: We have corrected the omission in the revised manuscript, and the\nfigure is now properly introduced as Supplementary Fig. 7 (Page 13 in main text).\nReviewer #3 (Remarks to the Author):\nThe manuscript “ZFP36L2, an interferon beta-induced inhibitor, restricts the nuclear\nexport of HIV-1 transcripts” by Pang et al. presents evidence for ZFP36L contributing to\nthe antiviral, IFN-beta-mediated response to HIV-1 infection. Other viruses inhibited by\nthis factor include HIV-2, SIV, EIAV, but not FIV and MLV. This is shown by knockout in\nthe context of IFN-beta treatment, and heterologous expression experiments in cell lines\nand in CD4+ T-cells from PLHIV. In vivo ZFP36L2 mRNA levels inversely correlated\nwith viremia. ZFP36L2 binds to Rev and interferes with nuclear export of unspliced and\npartially spliced HIV-1 mRNAs. They mapped the Rev-binding site within ZFB36L2 and\nvice versa. Together, I believe this is an exciting and carefully executed study.\nMajor concerns: I don’ t have major concerns and I commend the authors to this\nbeautiful and extensive study.\nMinor concerns:\n-not an essential requirement, but the reader wonders what made the authors start\nstudying ZFP36L2. Some sort of explanation would benefit the start of the flow of the\npaper.\nResponse 1: Thank you for this suggestion; we are happy to address it. We believe\nthat we have already provided the rationale for studying ZFP36L2 in the manuscript.\nSpecifically, we stated: “Furthermore, ZFP36 directly inhibits HIV-1 production by\nbinding to genomic RNA 34, while ZFP36L1 inhibits influenza A virus replication through\ntranslational repression by selectively targeting viral RNA 35” (Page 4, Lines 82–85), and\n“……..given that ZFP36 inhibits HIV-1 production by binding to viral genomic RNA….…”\n(Page 5, Line 105).\n-the discussion is in wide parts a reiteration of the results. Instead of repeating results,\nresults should be contextualised.\nResponse 2: Thank you for this valuable suggestion. To address this point, we have\nrevised the Discussion to reduce repetition of the Results and to better contextualize our\nfindings. In addition, we have incorporated relevant hypotheses to strengthen the\ninterpretative depth of the Discussion in this revised version. Please also refer to\nResponse 11 to Reviewer-2.\n-Extended Fig. 1a) “At 48 h post-transfection, HIV-1 capsids in culture were quantified\nusing p24 ELISA”. I don’ t think capsids are quantified, it’ s rather the mass of capsid\nprotein.\nResponse 3: We have revised the sentence accordingly: “At 48 h post-transfection,\nHIV-1 capsid protein in culture was quantified using p24 ELISA” in the revised\nmanuscript (Page 40, Line 919, 923; Page 48, Line 1110; Page 54, Line 1254).\n-Extended Fig. 1 c-e, “Jurkat cells were infected with 100 ng of HIV-1NL4-3”. I believe\ncells weren’ t infected with 100 ng of HIV-1, rather 100 ng of p24 equivalents.\nResponse 4: We have corrected the text throughout the manuscript to read “100 ng\np24-equivalent HIV-1” in place of “100 ng of HIV-1” in Supplementary Fig. 1c–e and\nother figures (Page 40, Line 926; Page 45, Line 1026; Page 46, Lines 1049, 1055; Page\n48, Line 1113; Page 55, Lines 1269; Page 56, 1281).\n-I acknowledge the nuclear/cytoplasmic separation was validated at the protein level\n(Fig. 2c), however apparently not at the RNA level (Fig. 2b). Showing purity of the\nnuclear and cytoplasmic compartment by amplifying a strictly nuclear and almost\nexclusively cytoplasmically located RNAs would be beneficial.\nResponse 5: Thank you for this recommendation. Following your suggestion, we\nperformed RT-PCR to assess the nuclear-enriched U6 snRNA. As shown in the new\nFig. 2c (right) and Fig. 2i, the separation of nuclear and cytoplasmic fractions was\nvalidated at the RNA level by measuring U6 snRNA. We have incorporated this\nclarification into the revised manuscript (Pages 8–9, Lines 184–187; Page 10, Line 214;\nPage 42, Lines 958–960, 972–973).\n-Immunoblots shown in Fig. 3 and extended Fig. 3 seem to imply that SFP36L2 is\nlocated both in the cytoplasm and nucleus. Does it have an NLS?\nResponse 6: Thank you for this question, and we are glad to address it.\nZFP36L2 is known to shuttle between the nucleus and the cytoplasm, as previously\nreported (PMID: 34611029; PMID: 16508015; PMID: 7958838). Consistent with this, our\ndata show that ZFP36L2 localizes to both compartments (Fig. 2c,h; Fig. 3c,e,i;\nSupplementary Fig. 3c,f), supporting its role as a nucleocytoplasmic shuttling protein.\nSimilar to rat ZFP36—which contains both a nuclear export signal (NES) and a\nnuclear localization signal (NLS), with the latter residing within the tandem zinc-finger\nmotifs (PMID: 12054509)—human ZFP36L2 also harbors an NES and an NLS, as\npreviously described (PMID: 11796723). Thus, members of the ZFP36 family contain\nboth NES and NLS sequences that mediate their shuttling between the nucleus and the\ncytoplasm (PMID: 11796723). Please also see our Response 3 to Reviewer 2.\n-the finding of the delta109–116 Rev mutant evading ZFP36L2 inhibition suggests HV-1\ncould evolve to evade restriction while maintaining a functional Rev – can this be\nrecapitulated in virus passaging experiments in ZFP36L2-overexpressing cells? Looking\nat different subtypes, is the 109-116 Rev region polymorphic?\nResponse 7: Thank you for this thoughtful question.\nAs shown in Fig. 4f–i (Extended Data Fig. 8f–i in the previous submission), we have\nalready performed virus passaging experiments demonstrating that the Δ109–116 Rev\nHIV-1 mutant is resistant to ZFP36L2-mediated restriction. Moreover, ZFP36L2 fails to\nbind the transcripts of the Δ109–116 Rev mutant while retaining binding to wild-type\nHIV-1 transcripts (Supplementary Fig. 7c,d).\nRegarding the polymorphism of the 109–116 region of Rev, we analyzed patient-\nderived HIV-1 Rev sequences from the Los Alamos HIV Sequence Database using the\n2022 curated group M Rev protein alignment, which contains 5,901 sequences. Within\nthis region, 5,305 of 5,901 sequences (89.9%) encode the full eight-residue motif, 149\nsequences (2.5%) contain partial deletions or premature stop codons, and 447\nsequences (7.6%) lack the entire segment. The consensus sequence across patient\nisolates is VLGSGTKE. Residues 110, 113, and 115 are highly conserved (L, G, and K\nat 99.7%, 92.9%, and 86.1%, respectively), whereas positions 109, 111, 112, 114, and\n116 show modest variability. Please also refer to Response 10 to Reviewer 2 for\nadditional detail.\nLines 403-404: “ZFP36L2 transcript levels were inversely correlated with plasma HIV-1\nRNA levels (Fig. 5b), supporting its role as a negative regulator of viral replication.”\nAlthough a causal link is a tempting hypothesis, I think it is more likely (or also likely)\nthat ZFP36L2 expression is merely a surrogate of ISG expression in general during\nunsuppressed HIV-1 infection. In other words, one would expect to see increased\nexpression of any ISG, antiviral or not, in untreated PLHIV. I suggest to rephrase more\ncarefully.\nResponse 8: Thank you for this valuable suggestion. Following your recommendation,\nwe have revised the sentence to read: “ZFP36L2 transcript levels were inversely\ncorrelated with plasma HIV-1 RNA levels (Fig. 6b). This correlation may not be specific,\nas ZFP36L2 is an IFN-stimulated gene, and similar correlations may also be observed\nfor other IFN-induced genes” in this revision (Page 18, Lines 399-402). Please also\nrefer to Response-4 to reviewer 1.\n-the experiments shown in Fig. 5 g (reconstitution of ZFP36L2 in primary cells) don’ t\nexclude the possibility that the factor was expressed at aberrantly high levels and that\nthis high overexpression is causing some antiviral state per se. It would have been nice\nto include some specificity control, like expressing the ZFP36L2 mutant that is no longer\nable to bind to Rev, as a negative control (instead of GFP)\nResponse 9: We followed your recommendation and performed new experiments using\nthe ZFP36L2 (Δ446–480) mutant, which fails to bind Rev. As shown in the new Fig. 6f–\nh in this revision, this mutant consistently did not affect HIV-1 spread in CD4⁺ T cells ex\nvivo. We have also updated the patient backgrounds in Supplementary Table 3.\nThe methods seem to lack a chapter about the lentiviral transduction of different cell\ntypes, especially primary T-cells and macrophages.\nResponse 10: Thank you for pointing this out. The procedures for lentiviral transduction\nin primary cells, including CD4⁺ T cells and macrophages, are well-established in our\nlaboratory. We have cited our previously published papers that describe these methods\nin detail (Refs. 40, 41, 46) and have now added the relevant descriptions to the\nMethods section (Pages 30–32, Lines 704–732). Please also see our Response-8 to\nReviewer 2."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments well. I have no further concerns. Interesting paper.", "reviewer_2": "The authors were very responsive and addressed all my comments, either by new experiments or textual changes. The result that ZFP36L2 is decreased during T cell activation is highly interesting suggesting that ZFP36L2 may be one of the factors that interferes with productive infection of resting T cells.\nI am still puzzled by the fact that knocking-down ZFP36L2 rescues approx 80% of the suppression imposed by IFNb. Even though the authors argue that this is not complete recovery and for instance MX2 may still restrict viral replication it is very suprising that all the other ISGs play a minor role here. But the data looks solid and is well controlled for. The short sentence added to the discussion does not really address this issue.\nRelated to the discussion, even given that two reviewers critisized the superficial appearance of the discussion the few modifications imposed by the authors do not really eliminate this shortcoming. However I consider this a minor point that does not impact on the overall data and conclusion.", "reviewer_3": "I am satisfied by the changes and comments addressing my (minor) concerns."}, "author_response": "Responses to Reviewers’ Comments\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my comments well. I have no further concerns. Interesting\npaper.\nResponse: We thank you for your valuable suggestions.\nReviewer #2 (Remarks to the Author):\nThe authors were very responsive and addressed all my comments, either by new\nexperiments or textual changes. The result that ZFP36L2 is decreased during T cell\nactivation is highly interesting suggesting that ZFP36L2 may be one of the factors that\ninterferes with productive infection of resting T cells.\nI am still puzzled by the fact that knocking-down ZFP36L2 rescues approx 80% of the\nsuppression imposed by IFNb. Even though the authors argue that this is not complete\nrecovery and for instance MX2 may still restrict viral replication it is very suprising that\nall the other ISGs play a minor role here. But the data looks solid and is well controlled\nfor. The short sentence added to the discussion does not really address this issue.\nRelated to the discussion, even given that two reviewers critisized the superficial\nappearance of the discussion the few modifications imposed by the authors do not\nreally eliminate this shortcoming. However I consider this a minor point that does not\nimpact on the overall data and conclusion.\nResponse: We sincerely thank the reviewer for the thoughtful feedback and for\nacknowledging the rigor and solidity of our data. We also appreciate the opportunity to\nclarify the intriguing observation that ZFP36L2 knockdown rescues approximately 80%\nof IFN-β–mediated suppression.\nThe reviewer correctly highlights a key question regarding the apparently minimal\ncontribution of other interferon-stimulated genes (ISGs). We agree that, in a\nphysiological context, IFN-β exerts its antiviral effects through a coordinated network of\nISGs. However, our experimental systems were intentionally designed to dissect the\nspecific contribution of ZFP36L2, which likely explains the observed high rescue\npercentage:\n1. Targeted Experimental Design: As detailed previously, we employed:\n(1) Proviral DNA transfection/electroporation, which bypasses early steps of viral\ninfection such as entry and reverse transcription, thereby reducing the impact of early-\nphase inhibitors like MX2, APOBEC3G, and related factors. (2) Infection timed with\npeak ZFP36L2 expression, when MX2 levels are declining.\nThis design effectively minimized the activity of major known HIV-restrictive\nISGs in our assays, allowing the role of ZFP36L2 to be revealed with clarity.\nImportantly, even under these conditions, ZFP36L2 silencing only rescues ~80% of IFN-\nβ–mediated suppression. The remaining ~20% likely reflects residual antiviral activity\nfrom other ISGs that cannot be entirely eliminated in our system (e.g., subtle effects of\nIFITMs on membrane composition or other uncharacterized factors), consistent with the\nmultifaceted nature of the interferon response.\nWe have now explicitly described this rationale in the Discussion to provide full\ncontext and transparency regarding the contribution of ZFP36L2 relative to other ISGs\n(page 21, lines 495–503). This ensures that the interpretation of the result is clear and\nplaced within the broader framework of ISG network biology.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed my comments well. I have no further concerns. Interesting\npaper.\nResponse: Thank you for your encouraging assessment."}]

Decision letter