Zinc finger protein ZNF33B facilitates Japanese encephalitis virus replication by regulating m6A modification on Ifih1 and Irf3 transcripts to antagonize antiviral response

Jian Du; Chunwei Li; Jinyan Zhang; Jiyuan Luo; Huizhi Zhang; Huanchun Chen; Xiangmin Li; Ping Qian

doi:10.1186/s13578-025-01526-3

. 2026 Jan 4;16:13. doi: 10.1186/s13578-025-01526-3

Zinc finger protein ZNF33B facilitates Japanese encephalitis virus replication by regulating m⁶A modification on Ifih1 and Irf3 transcripts to antagonize antiviral response

Jian Du ^1,^2,⁴, Chunwei Li ^1,^2,⁴, Jinyan Zhang ^1,^2,⁴, Jiyuan Luo ^1,^2,⁴, Huizhi Zhang ^1,^2,⁴, Huanchun Chen ^1,^2,⁴, Xiangmin Li ^1,^2,^3,^4,^✉, Ping Qian ^1,^2,^3,^4,^✉

PMCID: PMC12865994 PMID: 41486161

Abstract

Japanese encephalitis virus (JEV), a neurotropic flavivirus, poses a significant public health threat, yet the molecular mechanisms underlying its interaction with host immunity remain poorly understood. This study reveals that zinc finger protein ZNF33B promotes JEV replication by subverting the RLR-mediated innate immune response through orchestrating m⁶A RNA modification. ZNF33B directly binds to antiviral transcripts Ifih1 (encoding MDA5), Mavs, and Irf3, recruiting the m⁶A methyltransferase METTL14 to enhance their m⁶A methylation. Concurrently, ZNF33B interacts with the nuclear m⁶A reader YTHDC1 to facilitate the export of these methylated transcripts from the nucleus to the cytoplasm. In the cytoplasm, the m⁶A-modified transcripts are recognized by the cytoplasmic reader YTHDF2, leading to accelerated RNA decay. This process downregulates MDA5 and IRF3 protein levels, suppressing type I interferon production and downstream antiviral responses, thereby creating a permissive environment for JEV replication. Our findings establish a regulatory axis where ZNF33B integrates m⁶A modification and RNA metabolism to evade host immunity, highlighting the potential of targeting epitranscriptomic pathways for antiviral therapy.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13578-025-01526-3.

Introduction

Japanese encephalitis virus (JEV), a member of the Flaviviridae family and Flavivirus genus, poses a significant threat to global public health as a neurotropic pathogen(1, 2). It is transmitted primarily through the bite of infected Culex mosquitoes and is prevalent across Asia and the western Pacific, with an estimated 68,000 clinical cases reported annually(3). JEV infection can lead to severe neurological complications, including encephalitis, which results in mortality rates ranging from 10% to 30%(4). Among survivors, approximately 30%-50% suffer from permanent neurological sequelae, such as parkinsonism, seizures, and cognitive impairments(5). Despite its significant impact on human health, our understanding of the molecular mechanisms governing JEV replication and its interaction with host immune responses remains limited.

The innate immune response serves as the first line of defense against viral infections and is critical for restricting viral replication and spread(6). RIG-I-like receptors (RLRs), including RIG-I and MDA5, are cytoplasmic sensors that recognize viral RNA and initiate signaling cascades leading to the production of type I interferons (IFNs) and other antiviral effectors(7, 8). Upon viral infection, RLRs undergo conformational changes, allowing them to interact with mitochondrial antiviral signaling protein (MAVS)(9). This interaction triggers a series of downstream signaling events involving TBK1 and IRF3, ultimately leading to the transcriptional activation of IFN-β and other interferon-stimulated genes (ISGs). These IFNs then bind to cell surface receptors, activating the JAK-STAT pathway and amplifying the antiviral response(10). However, viruses have evolved various strategies to evade or suppress these host defense mechanisms. For instance, flaviviruses are known to degrade RIG-I via the proteasomal pathway or cleave MAVS to disrupt RLR signaling, thereby promoting viral replication(11-13).

Zinc finger proteins (ZNFs), characterized by their zinc finger domains, are one of the largest families of transcription factors in the human genome(14). Traditionally, they are recognized for their roles in DNA binding and transcriptional regulation(15). However, emerging evidence suggests that certain ZNFs can also interact with RNA, lipids, and other macromolecules, participating in diverse cellular processes such as RNA metabolism, protein translation, and post-translational modifications(16). Recently, a new characterized zinc finger protein ZNF33B has garnered attention due to its potential involvement in the regulation of embryonic development, organogenesis, cell differentiation, and viral infection (17-20). However, its specific role in viral infections, particularly in the context of JEV, remains to be elucidated. ZNF33B exhibits distinct expression profiles across diverse tissues. Notably, elevated expression levels of ZNF33B were observed in neural tissues, with particularly pronounced expression detected in the cerebral cortex and hippocampus. Hence, we hypothesized that ZNF33B might participate in neurotropic virus JEV replication.

N6-methyladenosine (m⁶A), the most abundant internal chemical modification in eukaryotic mRNA, plays a pivotal role in regulating various aspects of mRNA metabolism, including transcription, splicing, nuclear export, stability, and translation(21-23). m⁶A modification is dynamically regulated by a set of enzymes, including writers (e.g., METTL3 and METTL14), erasers (e.g., FTO and ALKBH5), and readers (e.g., YTHDF proteins, YTHDC1, and IGF2BP proteins)(24-26). These m⁶A-modifying enzymes recognize specific RNA sequences and structures, thereby influencing the fate of target mRNAs. Notably, m⁶A modifications have been implicated in the regulation of antiviral innate immunity. For example, hepatitis C virus (HCV) exploits m⁶A reader protein YTHDC2 to enhance viral IRES-dependent RNA translation, while Zika virus (ZIKV) manipulates m⁶A modifications to evade immune detection(27-29). However, the role of m⁶A modifications in JEV infection and the potential involvement of ZNF33B in this process have not been thoroughly investigated.

In this study, we aim to explore the role of ZNF33B in JEV replication and its underlying molecular mechanisms. We hypothesize that ZNF33B may modulate the innate immune response to facilitate JEV replication by interacting with key components of the RLR signaling pathway and influencing the m⁶A modification of antiviral transcripts. Our findings reveal that ZNF33B acts as a negative regulator of the RLR-mediated innate immune response by binding to antiviral transcripts such as Ifih1, Mavs, and Irf3, recruiting METTL14 to enhance their m⁶A modification, and collaborating with YTHDC1 to promote their nuclear export. Once in the cytoplasm, these m⁶A-modified transcripts are recognized by YTHDF2, leading to accelerated RNA decay. This process results in the downregulation of MDA5, MAVS, and IRF3 proteins, impairing the production of type I IFNs and downstream antiviral effectors, thereby creating a permissive environment for JEV replication. Our study highlights the potential of targeting epitranscriptomic pathways as a therapeutic strategy against viral infections.

Materials and methods

Virus and cell culture

JEV strain P3 was kindly provided by Professor Yi-ling Lin (Institute of Biomedical Sciences, Academia Sinica, Taiwan, China) and propagated in baby hamster kidney (BHK-21) cells. Human embryonic kidney HEK293T (HEK293T) cells and swine kidney-6 (SK6) cells were saved in our laboratory. Porcine kidney-15 (PK-15) Cas9 and ZNF33B KO cells were kindly provided by Professor Shuhong Zhao and Professor Shengsong Xie (Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, China). All cells were incubated in Dulbecco's modified Eagle's minimal essential medium (DMEM; Invitrogen, USA) containing 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin (GENVIEW), and 10 μg/mL streptomycin sulfate (GENVIEW) at 37℃ in a humidified 5% CO₂ incubator.

Plasmids

cDNA encoding full-length NS3 and NS5 from JEV RP9 strain was cloned into lentiviral-based vector pTRIP-3Flag-RFP, pEGFP-C1, pCAGGS-HA or pCR3.1-Flag as described previously. The open reading frames of specific host genes were subcloned into plasmids with epitope tags: ZNF33B-Flag, ZNF33B-HA, RIG-I-Flag, MDA5-Flag, MAVS-Flag, TBK1-Flag, IRF3-Flag, METTL3-Flag, METTL14-Flag, YTHDC1-HA, and YTHDF2-HA.

Reagents and antibodies

Actinomycin D (ActD) and DMSO were purchased from MCE (NY, USA). The antibodies probed by JEV NS3 (Cat. # GTX125868) were obtained from GeneTex (USA). The antibodies probed by GAPDH (Cat. # 60004-1-Ig), IgG (Cat. # 30000-0-AP), FLAG (Cat. # 20543-1-AP), HA (Cat. # 51064-2-AP, 66006-2-Ig), RIG-I (Cat. # 20566-1-AP), MDA5 (Cat. # 21775-1-AP), TBK1 (Cat. # 283971-1-AP), METTL14 (Cat. # 26158-1-AP), YTHDC1 (Cat. # 14392-1-AP), YTHDF2 (Cat. # 24744-1-AP) and m6A (Cat. # 68055-1-Ig) were obtained from Proteintech (USA). The antibodies probed by MAVS (Cat. # sc-166583) were obtained from Santa Cruz Biotechnology (USA). The antibodies probed by p-TBK1 (Cat. # 5483) and p-STAT1 (Cat. # 9167) were obtained from Cell Signaling Technology (USA). The antibodies probed by HA (Cat. # 18850), FLAG (Cat. # M185-6), and V5 (Cat. # M167-3) were obtained from MBL (Japan). The antibodies probed by dsRNA antibody (J2) (Cat. # 10010500) were obtained from Nordic MUbio (Netherlands). HRP-conjugated goat anti-mouse (Cat. # 330) and HRP-conjugated goat anti-rabbit (Cat. # 458) IgG (H+L) secondary antibodies were purchased from MBL (Japan). Alexa Fluor Plus 647 Goat anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody (Cat. # A32733), Alexa Fluor 488 goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody (Cat. # A32731), and Alexa Fluor 568 goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody (Cat. # A11011) were purchased from Invitrogen (USA).

Western blotting

The cells were lysed by NP40 lysis buffer (1% NP40, 1.19% HEPES, 0.88% NaCl, 0.04% EDTA) on ice for 2 h. After centrifugation at 12 000 rpm for 10 min, the protein supernatant was collected and mixed with SDS-PAGE protein loading buffer. The protein supernatant was then loaded into a 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by electrophoresis and wet transfer to polyvinylidene fluoride (PVDF) membranes (Roche, UK). Thereafter, the membranes were blocked and incubated with antibodies. The image was developed using Bio-Rad ChemiDoc XRS+ instrument and Image Lab software.

Immunoprecipitation (IP)

The prepared protein supernatant was incubated with an appropriate amount of antibody and Protein A+G magnetic beads (Beyotime, China) at 4 ℃ in a rolling incubator for 8-12 h. Subsequently, the mixture of protein samples and magnetic beads was subjected to magnetic separation, followed by five washes with ice-cold NP40 lysis buffer for 10 minutes each. The elution of proteins was boiled at 95 °C in 2× loading buffer for 10 minutes. The obtained proteins were utilized for Western blotting assay.

Immunofluorescence (IF)

After the implementation of appropriate treatment, cells cultured on glass coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.2% Triton X-100, and blocked with 5% bovine serum albumin (BSA). Then the cells were probed with primary antibodies, followed by exposure to fluorescent-dye-conjugated secondary antibodies. DAPI (Sigma-Aldrich, USA) was applied for nuclei staining. The images were visualized by STochastic optical reconstruction microscopy (Nikon, Japan).

Dual luciferase reporter assay

In the present investigation, HEK293T cells were initially seeded into 24-well plates. Subsequently, transfection procedures were performed using the pCAGGS-ZNF33B-HA plasmids, IFN-β-Luc reporters, and pRL-TK, facilitated by jetPRIME^® transfection reagent. Twenty-four hours post-transfection, cells were infected with JEV or SeV for 36 h. The enzymatic activities of firefly luciferase and Renilla luciferase were quantified in line with the manufacturer's protocol of the dual-luciferase reporter assay kit (Promega, Beijing, China). The results were obtained by normalizing the luciferase values against the internal control Renilla luciferase activity.

Viral titration

BHK-21 cell monolayers were incubated with serial dilutions of the cultured supernatant at 37℃ for 2 h, followed by being overlaid with DMEM containing 2% FBS and 3% carboxymethyl cellulose (CMC). After 4 days of incubation, viral plaques were stained with 1% crystal violet dye and counted for analysis.

Quantitative PCR (qPCR)

Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcribed into cDNA with HiScript 1st Strand cDNA Synthesis Kit (Vazyme, China) following the manufacturer's protocol. Quantitative real-time PCR analysis was performed using the ABI StepOne Plus system (Applied Biosystems) and qPCR SYBR Green Master Mix (Yeasen, China). Specific primers are provided in Supplementary Table 3. The relative mRNA expression was calculated by normalizing to GAPDH.

mRNA stability

The mRNA transcription was halted by exposure to 5 μg/mL of actinomycin D (ActD) (MCE, USA). Subsequently, cells were sampled at the preconfigured time for qPCR analysis. The mRNA decay rate was measured by non-linear regression curve fitting.

RNA interference

siRNAs targeting human METTL14, YTHDC1, and YTHDF2 were synthesized by Sangon Biotech (China) and delivered by jetPRIME^® transfection reagent (polyplus, USA) according to the manufacturer's short protocol. The sequences are displayed in Supplementary Table 2.

RNA immunoprecipitation (RIP)

For RIP analysis, a fraction of the cell lysate was set aside as input, while the remaining lysates were aliquoted and incubated overnight with anti-FLAG or control IgG antibodies pre-coated with Protein A+G magnetic beads (Beyotime, China) at 4 ℃ with rotation. Subsequently, the pellets were washed using ice-cold NT2 buffer. The immunoprecipitated samples were then subjected to Proteinase K treatment (Thermo Fisher Scientific, USA) at 55 ℃ for 20 min. The supernatant was collected for RNA purification and RT-PCR or qPCR analysis.

MeRIP-qPCR

Total RNA was extracted using Trizol reagent (Invitrogen, USA). Poly(A) RNA was specifically isolated using oligo(dT) magnetic beads (Thermo, USA). Fragmented RNA was pre-incubated with a Dynabeads Antibody Coupling Kit (Thermo, USA), and an m⁶A Ab (Proteintech, China) capable of recognizing m⁶A was utilized. The relative abundance of mRNA within the m⁶A RIP complexes derived from either JEV-infected cells or noninfected cells was assessed via RT-qPCR.

Cellular fraction

Nuclear and cytoplasmic compartments were separated using the Cytoplasmic&Nuclear RNA kit (Norgen Biotek Corp., Thorold, ON, Canada) according to the manufacturer’s instructions. The efficacy of cellular fractionation was validated by employing cytoplasmic marker Gapdh and nuclear marker Rnu6 as control indicators, respectively.

Statistical analysis

The data were displayed as means ± standard deviation (SD), and statistical comparisons were conducted by ANOVA and two-tailed unpaired Student’s t-test using GraphPad Prism 9 software. The p-value less than 0.05 was regarded as statistically significant.

Results

ZNF33B negatively regulates the RLR-mediated innate immune response

Studies have proven that phosphate-containing viral RNA in the cytoplasm and long dsRNA can be sensed by RIG-I-like receptors (RLRs), eliciting a fierce innate immune cascade to combat flavivirus infection. Therefore, we pondered whether ZNF33B could subvert innate immunity to promote JEV replication. First, we verified whether JEV RNA can be sensed by RLRs by assessing the interaction of RIG-I and MDA5 with JEV RNA in the JEV-infected cells. HEK293T cells were transfected with RIG-I-Flag or MDA5-Flag plasmids, followed by JEV infection. The prepared cell lysates were then subjected to an in vitro RNA-Immunoprecipitation (RIP) assay using an anti-Flag antibody. The results demonstrated that the 3’ untranslated region (3’UTR) of JEV RNA was successfully amplified in the immunoprecipitated samples of both RIG-I and MDA5 (Fig. 1A). We also repeated the RNA Immunoprecipitation (RIP) experiment and performed RIP-qPCR analysis to quantitatively measure the enrichment of JEV RNA in the immunoprecipitates. The data clearly show a significant enrichment of JEV RNA pulled down by both RIG-I and MDA5 compared to the IgG control (Fig. 1B). Furthermore, to provide direct visual and spatial evidence of this critical interaction, we performed confocal microscopy to assess the colocalization of RIG-I and MDA5 with JEV double-stranded RNA (dsRNA). The immunofluorescence staining assay revealed abundant signals and enrichment of JEV dsRNA-RLRs complex in the cytoplasm (Fig. S1). These findings strongly suggest that JEV RNA can be recognized by RLRs, thereby indicating its potential role in triggering the RLR-mediated innate immune response. Additionally, we found that JEV infection inhibited the expressions of RIG-I, MDA5, and MAVS to evade innate immunity (Fig. S2A). To elucidate whether ZNF33B impairs the RLR signaling pathway to block IFN-β production, HEK293T cells were transfected with expression plasmids for ZNF33B-HA and Flag-tagged signaling molecules (including RIG-I, MDA5, MAVS, TBK1, and IRF3). As expected, the results indicated that ZNF33B inhibits the expression of RIG-I, MAVS, TBK1, and IRF3, indicating that ZNF33B might subvert RLR signaling to promote JEV replication (Fig. 1C). Further, we validated the impact of ZNF33B on JEV replication and RLR signaling by transfecting HEK293T cells with the ZNF33B-Flag plasmid, followed by JEV infection for 48 hours. The findings revealed that ZNF33B augmented the protein level of JEV NS3, indicating that ZNF33B exhibits proviral activity. Additionally, ZNF33B suppressed the protein levels of MDA5, MAVS, p-IRF3, and p-STAT1 (Fig. 1D, E). On the contrary, depletion of Znf33b led to a significant augmentation of the MDA5, MAVS, p-IRF3, and p-STAT1 protein levels during JEV infection (Fig. S2B, C). Subsequently, we employed qPCR analysis to investigate the impact of ZNF33B on type I IFN signaling and antiviral immunity. The results revealed that overexpression of ZNF33B led to a reduction in the mRNA levels of Ifna2, Ifnb1, Isg15, and Isg56 (Fig. 1F). Conversely, depletion of Znf33b augmented the mRNA levels of Ifna2, Ifnb1, Isg15, and Isg56 (Fig. S1D). Concurrently, a dual luciferase reporter assay was conducted, demonstrating that ectopic expression of ZNF33B markedly inhibited the activation of the IFN-β promoter, irrespective of JEV or SeV infection (Fig. 1G). Collectively, these findings indicate that ZNF33B exerts a negative regulatory effect on the RLR-mediated innate immune response.

Fig. 1 — ZNF33B negatively regulates the RLR-mediated innate immune response. A RIP assay using anti-Flag antibody and RT-PCR analysis of the association between RIG-I and MDA5 protein and JEV RNA with JEV 3’-UTR specific primers in HEK293T cells transfected with Flag-tagged RIG-I and MDA5. B RIP assay using anti-Flag antibody and qPCR analysis of the association between RIG-I and MDA5 protein and JEV RNA with JEV 3’-UTR specific primers in HEK293T cells transfected with Flag-tagged RIG-I and MDA5. C Immunoblot analysis of the levels of ZNF33B, RIG-I, MDA5, MAVS, TBK1, and IRF3 protein in HEK293T cells transfected with ZNF33B-HA and Flag-tagged RIG-I, MDA5, MAVS, TBK1, and IRF3 for 36 h. D Immunoblot analysis of the levels of JEV NS3, RIG-I, MDA5, MAVS, p-IRF3, T-IRF3, p-TBK1, T-TBK1, p-STAT1, and T-STAT1 in HEK293T cells transfected with ZNF33B-Flag, followed by JEV infection for 48 h. E The data were statistically analyzed using One-way ANOVA compared to the EV group. E qPCR analysis of the mRNA levels of *Ifna2*, *Ifnb1*, *Isg15*, and *Isg56* in HEK293T cells transfected with ZNF33B-Flag, followed by JEV infection for 48 h. The data were statistically analyzed using Student’s t-test compared to the CON group. F Dual-luciferase analysis of IFN-β level in HEK293T cells transfected with ZNF33B-Flag, IFN-β-Luc reporters, and pRL-TK, followed by JEV or Sev infection for 36 h. The data were presented as the ratio between the activity of the reporter plasmid. All experiments were conducted in triplicate, and data are represented as mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001.

ZNF33B is associated with MDA5 and MAVS

To evaluate the interaction between ZNF33B and RLR-related targets, plasmids encoding ZNF33B-HA and Flag-tagged RIG-I, MDA5, MAVS, TBK1, and IRF3 were ectopically expressed in HEK293T cells. Cell lysates were then immunoprecipitated using an anti-Flag antibody. The results demonstrated that ZNF33B was co-immunoprecipitated in MDA5 and MAVS-transfected cells (Fig. S3A). Subsequently, transient transfection and co-immunoprecipitation experiments were performed to determine the interaction between ZNF33B and MDA5/MAVS by reciprocal Co-IP experiments. We observed a robust association between ZNF33B and MDA5/MAVS, irrespective of JEV infection (Fig. S3B-S3E). Besides, we transfected ZNF33B-MYC plasmids into cells and detected endogenous MDA5 and MAVS in ZNF33B-immunoprecipitated samples. The results also demonstrated that ZNF33B could interact with endogenous MDA5 and MAVS (Fig. S3F). Next, confocal microscopy was employed to investigate the colocalization of ZNF33B and MDA5/MAVS proteins. Immunofluorescence analysis revealed that ZNF33B and MDA5/MAVS proteins colocalized within the cytoplasm (Fig. S3G and S3H). The interaction between ZNF33B and MDA5 and MAVS led us to hypothesize whether ZNF33B promotes JEV replication by antagonizing MDA5 and MAVS. To test this hypothesis, HEK293T cells were transfected with ZNF33B-HA and MDA5-Flag or MAVS-Flag plasmids, followed by JEV infection for 48 h. The results demonstrated a reciprocal inhibitory effect between ZNF33B and MDA5 and MAVS. Moreover, the ectopic expression of ZNF33B alleviated the inhibition of JEV NS3 protein induced by MDA5 (Fig. S3I and S3J). These findings provide further evidence that ZNF33B exerts a negative regulatory effect on MDA5 and MAVS.

ZNF33B impels the nuclear export of antiviral transcripts to promote RNA decay

Zinc finger proteins constitute one of the most abundant protein families within the human genome. Although zinc finger domains (ZNFs) are predominantly recognized for their DNA-binding capabilities, emerging evidence indicates their ability to interact with RNA, lipids, methylated DNA, as well as proteins and post-translational modifications (PTMs) such as SUMOylation, ubiquitination, and methylation. To identify mRNAs that could potentially interact with ZNF33B, HEK293T cells were transfected with ZNF33B-Flag expression plasmids, followed by JEV infection for 36 h. Subsequently, RNA immunoprecipitation followed by quantitative polymerase chain reaction (RIP-qPCR) analysis was performed using anti-Flag or IgG antibodies. A significant enrichment of Ifih1, Mavs, and Irf3 mRNAs was observed in the immunoprecipitates obtained with the Flag antibody compared to the control IgG (Fig. 2A). Furthermore, the interaction between ZNF33B and these transcripts was determined by RIP-RT-PCR. The results demonstrated that ZNF33B is constitutively associated with Ifih1, Mavs, and Irf3 transcripts in JEV-infected cells (Fig. 2B). Mature mRNA is translocated from the nucleus to the cytoplasm, where it either undergoes translation or is subject to RNA decay processes. In order to explore the influence of ZNF33B on the subcellular distribution of antiviral transcripts, we investigated whether ZNF33B modulated the transport of these antiviral mRNAs from the nucleus to the cytoplasm. The distribution profiles of Rnu6 and Gapdh demonstrated efficient separation of cytoplasmic and nuclear fractions (Fig. 2C). In JEV-infected cells overexpressing ZNF33B, the nuclear abundance of Ifih1, Mavs and Irf3 transcripts was lower, while the cytoplasmic abundance of these transcripts was greater, relative to control cells, which suggested that ZNF33B impelled the nuclear export of these antiviral transcripts in the cytoplasm (Fig. 2D, S4A). Subsequently, we investigated the impact of ZNF33B on the protein levels of these antiviral proteins. As anticipated, overexpression of ZNF33B suppressed the MDA5, MAVS, TBK1, and IRF3 protein levels, implying that these antiviral mRNAs might undergo RNA degradation following their translocation into the cytoplasm (Fig. 2E-G, S4B and C). Consequently, we assessed the stability of antiviral transcripts via an RNA decay assay. The stability of Ifih1 and Irf3 transcripts, but not that of Mavs transcripts, was significantly increased in ZNF33B-deficient PK-15 cells infected with JEV after RNA synthesis was inhibited using actinomycin D (ActD), whereas a significant reduction in the stability of these transcripts was observed in ZNF33B-overexpressing cells (Fig. 2H-M). These findings strongly suggest that ZNF33B exerts a crucial role in RNA degradation of antiviral transcripts during viral infection.

Fig. 2 — ZNF33B impels the nuclear export of antiviral transcripts to promote RNA decay. A RIP assay using anti-Flag antibody and qPCR analysis of the association between ZNF33B protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with Flag-tagged ZNF33B, followed by JEV infection for 48 h. B RT-PCR analysis of the association between ZNF33B protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with Flag-tagged ZNF33B, followed by JEV infection for 48 h. C-D qPCR analysis of the extraction of cytoplasmic and nuclear RNA with *Rnu6*, *Gapdh*, *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* specific primers in HEK293T cells transfected with Flag-tagged ZNF33B, followed by JEV infection for 48 h. The efficiency of cellular fractionation was indicated by *Rnu6* and *Gapdh* as the nuclear and cytoplasmic controls, respectively. E Immunoblot analysis of the levels of MDA5 in HEK293T cells transfected with ZNF33B-HA and MDA5-Flag for 36 h. F Immunoblot analysis of the levels of MAVS in HEK293T cells transfected with ZNF33B-HA and MAVS-Flag for 36 h. G Immunoblot analysis of the levels of IRF3 in HEK293T cells transfected with ZNF33B-HA and IRF3-Flag for 36 h. H-J qPCR analysis of the level of *Ifih1*, *Mavs*, and *Irf3* in HEK293T cells transfected with ZNF33B-Flag followed by treatment with ActD for the indicated times. The half-life was calculated by nonlinear regression. K-M qPCR analysis of the level of *Ifih1*, *Mavs*, and *Irf3* in *Znf33b*^-/- PK15 cells followed by treatment with ActD for the indicated times. The half-life was calculated by nonlinear regression. All experiments were conducted in triplicate, and data are represented as mean ± SD. ^*p < 0.05 and ^***p < 0.001.

ZNF33B increases the m⁶A modification on antiviral transcripts

Previous studies have demonstrated that mRNA with pre-existing methylation enhances its export from the nucleus, a process mediated by binding to nuclear reader proteins. N⁶-methyladenosine (m⁶A) represents the most prevalent internal chemical modification in eukaryotic mRNA, playing a crucial role in fine-tuning various aspects of mRNA metabolism, including transcription, splicing, nuclear export, RNA stability, and translation. Firstly, we explored the impact of JEV infection on the overall cellular m6A level. The immunostaining results showed that JEV infection elevated the total m⁶A level (Fig. 3A). To investigate the presence of m⁶A modification on antiviral transcripts, we performed m⁶A-specific MeRIP-qPCR on RNA extracted from cells that were either mock-infected or infected with JEV. The results clearly show a significant, JEV infection-dependent increase in the m⁶A modification levels of the Ifih1, Mavs, and Irf3 transcripts (Fig. 3B). Subsequently, HEK293T cells were transfected with methyltransferase METTL3-HA or METTL14-HA expression plasmids, followed by infection with JEV for 48 h. RIP-qPCR analysis was performed using anti-HA or IgG antibodies. The results revealed a significant enrichment of Ifih1, Mavs, and Irf3 mRNA in the immunoprecipitates obtained with the HA antibody compared to the control IgG (Fig. 3C, D). To predict m⁶A modification sites on the mRNA sequences, the SRAMP tool (http://www.cuilab.cn/sramp/) was utilized. Notably, the m⁶A modification positions were identified with high confidence at c. 664 bp on Ifih1, c. 1202 bp on Mavs, and c. 443 bp on Irf3 (Fig. S5). Accumulating evidence has demonstrated that m⁶A modification is implicated in controlling antiviral innate immunity(28, 30, 31). In light of this, we investigated the effect of methyltransferase METTL3 on the protein expressions of MDA5, MAVS, and p-IRF3 in JEV-infected cells. Our findings demonstrated that the protein levels of MDA5, MAVS, and p-IRF3 were significantly inhibited by METTL3 (Fig. 3E-F). Concurrently, we posited that ZNF33B modulated innate immunity by affecting the RNA methylation of antiviral transcripts within the nucleus. In JEV-infected cells, following the overexpression of ZNF33B, transcripts bound by anti-METTL3 exhibited a higher degree of enrichment for Mavs and Irf3, yet not for Ddx58 and Tbk1, which are not METTL3 targets (Fig. 3G, H). This finding suggested that ZNF33B could regulate the m⁶A RNA methylation of antiviral transcripts in JEV-infected cells. Given that ZNF33B was found to not only bind directly to antiviral transcripts to govern their stability but also modulate m⁶A RNA methylation, we investigated whether ZNF33B interacted with m⁶A writer proteins. Intriguingly, we discovered that ZNF33B displayed a notable association with the adaptor protein METTL14, rather than the catalytic protein METTL3 (Fig. 3I, J). Subsequently, we compared the cytoplasmic and nuclear abundances of m⁶A-modified antiviral transcripts. In JEV-infected cells with METTL14 overexpression, the cytoplasmic abundances of Ifih1, Mavs, and Irf3 transcripts were significantly higher, while their nuclear abundances were lower, as compared to cells without METTL14 overexpression, suggesting that METTL14 promotes the nuclear export of antiviral transcripts to reduce their expression (Fig. S6A-J). To clarify whether METTL14 is the primary mediator of ZNF33B-dependent m⁶A modification, we determined the effect of METTL14 on the levels of these antiviral proteins. METTL14 overexpression reduced MDA5, MAVS, and p-IRF3 in JEV-infected cells, aligning with its direct interaction with ZNF33B (Fig. 3K). Next, we built METTL14 knockdown cells by transfecting siRNAs (Fig. 3L) to assess whether ZNF33B’s proviral activity is dependent on METTL14. Our data ZNF33B overexpression reduced MDA5 and p-IRF3 in siCtrl cells, but METTL14 knockdown rescued this inhibition (Fig. 3M). Taken together, our findings established that ZNF33B orchestrates RNA methylation through the recruitment of METTL14 to impede innate immunity after JEV infection.

Fig. 3 — ZNF33B increases the m⁶A modification on antiviral transcripts. A Confocal microscope observation of the m6A level in HEK293T cells transfected with ZNF33B-HA, followed by JEV infection. Scale bar, 5 μm. B MeRIP assay using anti-m⁶A antibody and qPCR analysis of the m6A level with *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* specific primers in HEK293T cells infected with JEV. C RIP assay using anti-HA antibody and qPCR analysis of the association between METTL3 protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with HA-tagged METTL3, followed by JEV infection for 48 h. D RIP assay using anti-HA antibody and qPCR analysis of the association between METTL14 protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with HA-tagged METTL14, followed by JEV infection for 48 h. E Immunoblot analysis of the levels of MDA5, MAVS, p-IRF3, and T-IRF3 in HEK293T cells transfected with METTL3-HA, followed by JEV infection for 48 h. F The data were statistically analyzed using One-way ANOVA compared to the EV group. G RIP assay using anti-HA antibody and qPCR analysis of the effect of ZNF33B on the association between METTL3 protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with HA-tagged METTL3 and Flag-tagged ZNF33B, followed by JEV infection for 48 h. H Immunoblot analysis of the expression of METTL3 and ZNF33B in HEK293T cells transfected with HA-tagged METTL3 and Flag-tagged ZNF33B, followed by JEV infection for 48 h. I-J Immunoblot analysis of the association of ZNF33B with m⁶A reader proteins by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-HA and Flag-tagged METTL3 or METTL14. The cell lysates were immunoprecipitated with anti-Flag antibody. K Immunoblot analysis of the levels of MDA5, MAVS, p-IRF3, and T-IRF3 in HEK293T cells transfected with METTL14-HA, followed by JEV infection for 48 h. L Immunoblot analysis of the levels of METTL14 in HEK293T cells transfected with METTL14 siRNAs. M Immunoblot analysis of the levels of MDA5, MAVS, p-IRF3, and T-IRF3 in HEK293T cells transfected with ZNF33B-HA and METTL14 siRNA, followed by JEV infection for 48 h. All experiments were conducted in triplicate, and data are represented as mean ± SD. ^*p < 0.05 and ^**p < 0.01.

ZNF33B recruits nuclear m⁶A reader YTHDC1 to impel the export of antiviral transcripts

The fate of m⁶A-modified mRNAs is deciphered by m⁶A readers, predominantly present in the cytoplasm, with the exception of the nuclear-localized YTHDC1. Given that YTHDC1 has been associated with nuclear export, transcription, splicing, and the stability of methylated transcripts in the nucleus, we investigated the mRNAs that could potentially be recognized by YTHDC1 in JEV-infected cells. Through RIP-qPCR assay using an HA antibody, a notable enrichment of Ifih1, Mavs, Traf3, and Ifna2 mRNAs was observed in the immunoprecipitates, as compared to the control IgG (Fig. 4A). Moreover, the cytoplasmic and nuclear RNA fractions were isolated to determine the effect of YTHDC1 on the nuclear export of antiviral transcripts. The results showed that YTHDC1 promoted the nuclear export of antiviral transcripts (Fig. 4B). Our prior research demonstrated that ZNF33B regulates RNA m⁶A modification and predominantly localizes within the nuclear compartment, which led us to the hypothesis that ZNF33B may interact with YTHDC1. First, we detected the effect of ZNF33B on the protein expression of YTHDC1. The results showed that ZNF33B increased the protein level of YTHDC1 (Fig. 4C). In HEK293T cells, we observed the reciprocal combination between ZNF33B and YTHDC1 (Fig. 4D, E). Then, confocal microscopy was employed to verify the subcellular localization of ZNF33B and YTHDC1. Intriguingly, ZNF33B was found to form distinct nuclear condensates with YTHDC1, potentially modulating RNA metabolism (Fig. 4F). Therefore, we hypothesized that YTHDC1 is required for ZNF33B-mediated nucleocytoplasmic transport of antiviral transcripts. We generated YTHDC1 Knockdown HEK293T cells via transfecting with 3 siRNAs (Fig. S7). Then, we compared the nucleocytoplasmic distribution of Ifih1, Mavs, and Irf3 transcripts in ZNF33B overexpressed cells with and without YTHDC1 knockdown during JEV infection. The efficiency of cellular fractionation was validated using Rnu6 (nuclear) and Gapdh (cytoplasmic) controls (Fig. 4G). ZNF33B overexpression reduced nuclear abundance of Ifih1, Mavs, and Irf3 while increasing cytoplasmic abundance. However, YTHDC1 knockdown rescued the nuclear abundance and reduced the cytoplasmic abundance of these transcripts induced by ZNF33B (Fig. 4H-J). In general, these findings indicated that ZNF33B could recruit YTHDC1 to impel the nuclear export of m⁶A-modified mRNAs.

Fig. 4 — ZNF33B recruits nuclear m6A reader YTHDC1 to impel the export of antiviral transcripts. A RIP assay using anti-HA antibody and qPCR analysis of the association between YTHDC1 protein and *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with HA-tagged YTHDC1, followed by JEV infection for 48 h. B qPCR analysis of the cellular distribution of *Ddx58*, *Ifih1*, *Mavs*, *Tbk1*, and *Irf3* with specific primers in HEK293T cells transfected with HA-tagged YTHDC1. C Immunoblot analysis of the levels of YTHDC1 in HEK293T cells transfected with ZNF33B-Flag and YTHDC1-HA for 48 h. D Immunoblot analysis of the association of ZNF33B with YTHDC1 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-Flag and YTHDC1-HA. The cell lysates were immunoprecipitated with anti-Flag antibody. E Immunoblot analysis of the association of ZNF33B with YTHDC1 by immunoprecipitation of lysates from HEK293T cells transfected with ZNF33B-Flag and YTHDC1-HA. The cell lysates were immunoprecipitated with anti-HA antibody. F Confocal microscope observation of the colocalization of ZNF33B with YTHDC1 in HEK293T cells transfected with ZNF33B-Flag and YTHDC1-HA. Scale bar, 10 μm. G-J qPCR analysis of the extraction of cytoplasmic and nuclear RNA with *Rnu6*, *Gapdh*, *Ifih1*, *Mavs*, and *Irf3* specific primers in HEK293T cells transfected with ZNF33B-Flag and YTHDC1 siRNA, followed by JEV infection for 48 h. The efficiency of cellular fractionation was indicated by *Rnu6* and *Gapdh* as the nuclear and cytoplasmic controls, respectively. All experiments were conducted in triplicate, and data are represented as mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001.

YTHDC1 facilitates JEV replication

Our previous investigation revealed that YTHDC1 promoted the nuclear export of antiviral transcripts. Nevertheless, the function of YTHDC1 in the replication of JEV remains elusive. Initially, we explored the impact of YTHDC1 on JEV replication in HEK293T cells. The results indicated that YTHDC1 augmented JEV replication by elevating the protein level of NS3 (Fig. 5A, B). Furthermore, supernatants from JEV-infected cells were collected at 48 h post-infection to determine viral titers using the plaque assay. Significantly, an elevation in viral loads was observed in cells expressing YTHDC1 (Fig. 5C, D). Additionally, cells were transfected with different doses of YTHDC1-HA expression plasmids, along with Flag-NS3 and NS5, for 24 h. We observed that YTHDC1 upregulated the levels of NS3 and NS5 proteins (Fig. 5E-H). Currently, the function of YTHDC1-mediated export of antiviral transcripts from the nucleus to the cytoplasm, whether for translation or RNA decay, remains unclear. Consequently, we determined the effect of YTHDC1 on the expression of antiviral proteins. YTHDC1 overexpression led to a significant reduction in the protein levels of MDA5, MAVS, and p-IRF3 (Fig. 5I). Taken together, our data revealed that YTHDC1 facilitates JEV replication through counteracting antiviral innate immunity.

Fig. 5 — YTHDC1 facilitates JEV replication. A Immunoblot analysis of the levels of JEV NS3 in HEK293T cells transfected with YTHDC1-HA, followed by JEV infection for 48 h. B The data were statistically analyzed using One-way ANOVA compared to the CON group. C The viral titration analysis of the supernatant in JEV-infected HEK293T cells expressing YTHDC1 was conducted by plaque assay. D The statistical analysis of JEV titer in YTHDC1-overexpressed cells using Student’s t-test compared to the CON group. E Immunoblot analysis of the levels of JEV NS3 in HEK293T cells transfected with JEV NS3-Flag and YTHDC1-HA for 36 h. F The data were statistically analyzed using One-way ANOVA compared to the CON group. G Immunoblot analysis of the levels of JEV NS5 in HEK293T cells transfected with JEV NS5-Flag and YTHDC1-HA for 36 h. H The data were statistically analyzed using One-way ANOVA compared to the CON group. I Immunoblot analysis of the levels of MDA5, MAVS, p-IRF3, and T-IRF3 in HEK293T cells transfected with YTHDC1-HA, followed by JEV infection for 48 h. All experiments were conducted in triplicate, and data are represented as mean ± SD. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001.

ZNF33B inhibited innate immunity by promoting RNA decay of antiviral transcripts in a YTHDF2-dependent manner

In light of YTHDF2's function as a reader within the m⁶A epigenetic marking system, where its decoding activity frequently leads to the accelerated degradation of target mRNAs, we posited that these antiviral transcripts are recognized by YTHDF2 for RNA decay following shuttling from the nucleus to the cytoplasm. Through the ectopic expression of YTHDF2, we observed a significant downregulation in the mRNA levels of Ifih1 and Irf3 (Fig. 6A). To further validate our hypothesis, an analysis of the binding between YTHDF2 and transcripts was conducted via RIP-qPCR. In JEV-infected cells, a notable association was detected between YTHDF2 and the transcripts of Ifih1, Mavs, and Irf3 (Fig. 6B). In parallel, upon the inhibition of RNA synthesis using ActD, the stability of Ifih1 and Irf3 transcripts was significantly reduced, while that of Mavs transcripts remained unaffected in YTHDF2-expressing cells infected with JEV (Fig. 6C-E). Since our data revealed that YTHDF2 accelerated the RNA decay of Ifih1 and Irf3 transcripts, we next detected the effect of YTHDF2 on the protein levels of MDA5, MAVS, and IRF3. The results showed that YTHDF2 impaired the exogenous expression of MDA5 and IRF3, not MAVS (Fig. 6F-H). Furthermore, the endogenous protein levels of MDA5 and p-IRF3 were also suppressed by YTHDF2 (Fig. 6I), suggesting that YTHDF2 promotes Ifih1 and Irf3 mRNA decay to inhibit their protein expression.

Besides, we performed co-immunoprecipitation experiments, showing a reciprocal interaction between ZNF33B and YTHDF2 (Fig. 6J and K). Furthermore, confocal microscopy confirms a significant colocalization between ZNF33B and YTHDF2 in the cellular context (Fig. 6L). Subsequently, we detected the effect of ZNF33B on the protein expression of YTHDF2. The results showed that ZNF33B increased the protein levels of YTHDF2 (Fig. 6M, S8A). Notably, in JEV-infected cells overexpressing ZNF33B, the association between YTHDF2 and transcripts encoding Ifih1, Mavs, and Irf3 was significantly enhanced. Conversely, the binding of the Ddx58 transcript to YTHDF2 remained unchanged (Fig. 6N, S9A). To prove YTHDF2 is required for ZNF33B-mediated RNA decay, we generated YTHDF2 knockdown HEK293T cells via transfecting with 3 siRNAs, and the results showed that the YTHDF2 protein was significantly decreased in KD cells (Fig. S9B). We observed that ZNF33B overexpression shortens Ifih1 half-life from 15.20 h to 4.68 h and Irf3 half-life from 13.43 h to 5.63 h, while YTHDF2 knockdown prolonged the Ifih1 (4.68 h to 7.92 h) or Irf3 (5.63 h to 8.47 h) half-lives (Fig. 6O, P). However, Mavs' half-life (less dependent on YTHDF2) remained unchanged (Fig. 6Q). These findings strongly suggest that ZNF33B modulates the stability of antiviral transcripts through an m⁶A-dependent mechanism, where YTHDF2 facilitates mRNA degradation (Fig. 7).

Fig. 7 — Model for the mechanism by which ZNF33B regulates m⁶A modification on *Ifih1* and *Irf3* transcripts to promote JEV replication. Upon JEV infection, ZNF33B is resident in the nucleus and enhances the m⁶A modification on *Ifih1* and *Irf3* transcripts by interacting with m⁶A methyltransferases METTL14. Subsequently, the m⁶A-modified *Ifih1* and *Irf3* transcripts were recognized by m⁶A-reading protein YTHDC1 to be transported into the cytoplasm and degraded by YTHDF2, ultimately resulting in the suppression of the antiviral immune response.

To further evaluate the role of YTHDF2 in JEV replication, HEK293T cells were transfected with YTHDF2-HA, followed by JEV infection. The results showed that the exogenous protein level of NS3 and NS5 was increased by YTHDF2 (Fig. S8B). Furthermore, YTHDF2 also enhanced the protein levels of JEV NS3 and NS5 (Fig. S8C, D). In summary, our findings indicated that ZNF33B inhibited innate immunity by promoting RNA decay of antiviral transcripts in a YTHDF2-dependent manner.

Discussion

The interplay between neurotropic viruses and host innate immunity is a central focus of viral pathogenesis research, as understanding these dynamic interactions provides critical insights for developing targeted antiviral strategies(32). Japanese encephalitis virus (JEV), a flavivirus with high neurovirulence, has evolved sophisticated mechanisms to evade host defenses, yet the epitranscriptomic regulatory networks underlying its immune escape remain largely unexplored. In this study, we identify zinc finger protein ZNF33B as a key viral facilitator that subverts the RLR-mediated innate immune response through orchestrating N6-methyladenosine (m⁶A) modification of antiviral transcripts, uncovering a novel cross-talk between zinc finger protein function and epitranscriptomic regulation in viral infection.

The RIG-I-like receptor (RLR) signaling pathway serves as the first line of cytoplasmic defense against RNA viruses, with MDA5 (encoded by Ifih1), MAVS, and IRF3 acting as core components that drive type I interferon (IFN) production. Flaviviruses have previously been shown to antagonize this pathway via strategies such as proteasomal degradation of RIG-I or cleavage of MAVS(13, 33, 34). Our findings extend this knowledge by demonstrating that JEV exploits host ZNF33B to inhibit the RLR pathway through a distinct mechanism: instead of directly targeting protein stability or cleavage, ZNF33B modulates the epitranscriptomic fate of key antiviral transcripts. Specifically, ZNF33B binds to Ifih1, Mavs, and Irf3 mRNAs, initiating a cascade of m⁶A modification, nuclear export, and cytoplasmic decay. This mode of regulation is particularly notable because it targets multiple nodes of the RLR pathway simultaneously, from RNA sensors (MDA5) to adaptors (MAVS) and transcription factors (IRF3), representing a more comprehensive immune suppression strategy compared to single-target mechanisms reported in other flaviviruses. Furthermore, the reciprocal inhibitory interaction between ZNF33B and MDA5/MAVS suggests a dynamic tug-of-war during infection, where ZNF33B tips the balance toward immune tolerance by dampening the expression of these critical antiviral proteins.

Recent studies have highlighted the RNA-binding capacity of certain ZNFs, such as ZNF598 in regulating ribosome-associated quality control(35, 36). Here, ZNF33B’s unique ability to target multiple components of the pathway—from RNA sensors (MDA5) to adaptors (MAVS) and transcription factors (IRF3)—suggests a comprehensive strategy to cripple the antiviral response. ZNF33B’s role as an RNA-binding protein with dual functions—direct transcriptional regulation and epitranscriptomic modulation—challenges the traditional view of zinc finger proteins as DNA-centric regulators. Furthermore, a groundbreaking aspect of this study lies in linking ZNF33B to m⁶A RNA modification. The m⁶A epitranscriptome has emerged as a critical regulator of RNA metabolism, influencing splicing, stability, and translation(37). Viruses often hijack host epigenetic and epitranscriptomic machinery to enhance their replication. HBV-induced O-GlcNAcylation in YTDHF2 preserved the stability of MCM2/5 transcripts to promote HBV-related HCC tumorigenesis, while YTHDC1 inhibits NS splicing by binding to an NS 3' splicing site and promotes IAV replication and pathogenicity(38, 39). Our data show that ZNF33B enhances m⁶A deposition on Ifih1, Mavs, and Irf3 transcripts via its association with METTL14, a component of the m⁶A methyltransferase complex. While METTL3 typically serves as the catalytic subunit, METTL14 stabilizes METTL3 and enhances substrate recognition. The preferential interaction of ZNF33B with METTL14, rather than METTL3, suggests a specialized role in directing methylation to specific antiviral RNAs.

The nuclear export of m⁶A-modified transcripts is a tightly regulated process often mediated by reader proteins. YTHDC1, the sole nuclear m⁶A reader, has been implicated in splicing and export of methylated RNAs(40, 41). Our work demonstrates that ZNF33B forms nuclear condensates with YTHDC1, facilitating the cytoplasmic translocation of antiviral transcripts. This observation aligns with studies showing that m⁶A-modified RNAs are preferentially exported to the cytoplasm for translation or decay(42). However, in the context of JEV infection, nuclear export does not culminate in productive translation but rather in YTHDF2-mediated degradation. YTHDF2, a cytoplasmic reader, binds m⁶A marks and recruits deadenylases to destabilize target mRNAs(43). Our data reveal that ZNF33B amplifies YTHDF2’s activity, creating a feedforward loop that ensures rapid turnover of antiviral transcripts. This dual reliance on nuclear and cytoplasmic readers underscores the spatial coordination required for efficient immune evasion. Taken together, these findings expand the repertoire of proteins that interface with the m⁶A machinery, highlighting ZNF33B as a novel adaptor bridging RNA-binding proteins and epigenetic modifiers.

In conclusion, our study uncovers a novel mechanism by which ZNF33B facilitates JEV replication through orchestrating m⁶A modification of antiviral transcripts, promoting their nuclear export and YTHDF2-dependent degradation, and ultimately suppressing the RLR-mediated innate immune response. This work expands our understanding of the roles of zinc finger proteins and epitranscriptomic regulation in viral immune escape, and provides a foundation for the development of targeted antiviral therapies (Figure. 7). These findings not only advance our understanding of flaviviral pathogenesis but also highlight the therapeutic potential of targeting epitranscriptomic pathways in combating viral infections.

Supplementary Information

Supplementary Material 1^{(23MB, docx)}

Acknowledgments

We express our sincere gratitude to Shuhong Zhao (Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University) for generously providing Znf33b^-/- PK-15 cells and Hongbo Zhou (State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University) for generously providing pCAGGS-YTHDC1-HA plasmids. We would like to thank the National Key Laboratory of Agricultural Microbiology Core Facility for assistance in Confocal microscopy.

Author contributions

J.D. performed the experiment, analyzed the data, and drafted the manuscript. C.L., J.Z., J.L., and H.Z. helped with the investigation. S.X. provided the materials. H.C., X.L., and Q.P. contributed to the experimental design, manuscript review and editing, provision of resources, acquisition of funding, and project supervision.

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers: 32072901, 32373046) and the National Key Laboratory of Agricultural Microbiology (AML2023B11).

Data availability

The primary data underpinning the findings of this study are accessible within the article and its Supplementary Information or can be obtained from the corresponding author upon request. Source data are provided in this paper.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Xiangmin Li, Email: lixiangmin@mail.hzau.edu.cn.

Ping Qian, Email: qianp@mail.hzau.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(23MB, docx)}

Data Availability Statement

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[CR28] 28.Denolly S, Stukalov A, Barayeu U, Rosinski AN, Kritsiligkou P, Joecks S, et al. Zika virus remodelled ER membranes contain proviral factors involved in redox and methylation pathways. Nat Commun. 2023;14:8045. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[CR34] 34.Li CX, Tang CY, Liu XQ, Liu Y, Zhang LJ, Shi J, et al. E3 ubiquitin ligase MARCH5 positively regulates Japanese encephalitis virus infection by catalyzing the K27-linked polyubiquitination of viral E protein and inhibiting MAVS-mediated type I interferon production. MBio. 2025;16:e0020825. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Miścicka A, Bulakhov AG, Kuroha K, Zinoviev A, Hellen C, Pestova T. Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC. Nucleic Acids Res. 2024;52:4627–43. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Geng J, Li SX, Li Y, Wu ZH, Bhurtel S, Rimal S, et al. Stalled translation by mitochondrial stress upregulates a CNOT4-ZNF598 ribosomal quality control pathway important for tissue homeostasis. Nat Commun. 2024;15:1637. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Boulias K, Greer EL. Biological roles of adenine methylation in RNA. Nat Rev Genet. 2023;24:143–60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Zhu YX, Wang RF, Zou JH, Tian S, Yu LY, Zhou YB, et al. N6-methyladenosine reader protein YTHDC1 regulates influenza A virus NS segment splicing and replication. PLoS Pathog. 2023;19:e1011305. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Yang Y, Yan Y, Yin JX, Tang N, Wang K, Huang LY, et al. O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N6-methyladenosine-dependent manner. Signal Transduct Target Ther. 2023;8:63. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Roundtree IA, Luo GZ, Zhang ZJ, Wang X, Zhou T, Cui YQ, et al. YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs. Elife. 2017;6:e31311. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR41] 41.Widagdo J, Anggono V, Wong JJL. The multifaceted effects of YTHDC1-mediated nuclear m6A recognition. Trends Genet. 2022;38:325–32. [DOI] [PubMed] [Google Scholar]

[CR42] 42.Zhong SX, Li XD, Li CH, Bai HY, Chen JJ, Gan L, et al. Serrate drives phase separation behaviours to regulate m6A modification and miRNA biogenesis. Nat Cell Biol. 2024;26:2129–43. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Wen JY, Xue L, Wei Y, Liang JN, Jia WL, Yong TY, et al. YTHDF2 is a therapeutic target for HCC by suppressing immune evasion and angiogenesis through ETV5/PD-L1/VEGFA axis. Adv Sci. 2024;11:e2307242. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Zinc finger protein ZNF33B facilitates Japanese encephalitis virus replication by regulating m6A modification on Ifih1 and Irf3 transcripts to antagonize antiviral response

Jian Du

Chunwei Li

Jinyan Zhang

Jiyuan Luo

Huizhi Zhang

Huanchun Chen

Xiangmin Li

Ping Qian

Abstract

Supplementary Information

Introduction

Materials and methods

Virus and cell culture

Plasmids

Reagents and antibodies

Western blotting

Immunoprecipitation (IP)

Immunofluorescence (IF)

Dual luciferase reporter assay

Viral titration

Quantitative PCR (qPCR)

mRNA stability

RNA interference

RNA immunoprecipitation (RIP)

MeRIP-qPCR

Cellular fraction

Statistical analysis

Results

ZNF33B negatively regulates the RLR-mediated innate immune response

Fig. 1.

ZNF33B is associated with MDA5 and MAVS

ZNF33B impels the nuclear export of antiviral transcripts to promote RNA decay

Fig. 2.

ZNF33B increases the m6A modification on antiviral transcripts

Fig. 3.

ZNF33B recruits nuclear m6A reader YTHDC1 to impel the export of antiviral transcripts

Fig. 4.

YTHDC1 facilitates JEV replication

Fig. 5.

ZNF33B inhibited innate immunity by promoting RNA decay of antiviral transcripts in a YTHDF2-dependent manner

Fig. 6.

Fig. 7.

Discussion

Supplementary Information

Acknowledgments

Author contributions

Funding

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Zinc finger protein ZNF33B facilitates Japanese encephalitis virus replication by regulating m⁶A modification on Ifih1 and Irf3 transcripts to antagonize antiviral response

ZNF33B increases the m⁶A modification on antiviral transcripts

ZNF33B recruits nuclear m⁶A reader YTHDC1 to impel the export of antiviral transcripts