Targeting DNA Methyltransferase 1 Limits Vesicular Stomatitis Virus Through Enhancing IRF3/IFN Signaling

Background Viruses develop strategies to escape from host anti-viral response in many aspects including hijacking host epigenetic factors. However, the relationship between host DNA methyltransferase 1 (DNMT1) and vesicular stomatitis virus (VSV) replication is largely unknown. In the study, we performed PCR and Cell to uncover the of in VSV: Vesicular Stomatitis Virus; DNMT1: DNA methyltransferase1; CGIs: CpG islands; dsRNA: Double-stranded RNA; IRF3: IFN-regulatory factor 3; ISGs: IFN-stimulated genes; ISREs: IFN-stimulated response elements; ISGF3: IFN-stimulated gene factor 3; STAT: signal transductor and activator of transcription; ERV: Endogenous retroviruses; shRNA: short hairpin RNA; MSP: Methylation-Specic PCR; MOI: infection.


Abstract Background
Viruses develop strategies to escape from host anti-viral response in many aspects including hijacking host epigenetic factors. However, the relationship between host DNA methyltransferase 1 (DNMT1) and vesicular stomatitis virus (VSV) replication is largely unknown.

Methods
In the present study, we performed Western Blotting, qRT-PCR, RNA interference, Methylation-speci c PCR and Cell viability assay to uncover the role of DNMT1 in the process of VSV replication.

Results
We have observed VSV infection enhances DNMT1 protein accumulation in macrophage cell. Furthermore, DNMT1 functional blocking and gene silencing limit VSV replication. Moreover, loss of DNMT1 increases interferon responses, including Ifnb1 and IFN-stimulated genes (ISGs) upregulation. CpG islands (CGIs) of Irf3 promoter region are demethylated after DNMT1 short-term inhibition with thioguanine, which is accompanied by Irf3 transcript upregulation. Meanwhile, Irf3 silencing largely reversed DNMT1 loss-suppressed VSV replication. What is more, the basal level of endogenous retrovirus (ERV) transcripts is required for thioguanine-induced ISGs.
Conclusions DNMT1 loss-induced IRF3 enhancement leads to interferon responses and subsequent VSV suppression, which provides a potential strategy to inhibit viral replication by targeting DNMT1 with its inhibitor.
After sensing of viruses, PRRs trigger downstream transcriptional cascades in chromatin level, which is mainly determined by epigenetic regulators [6]. Meanwhile, viruses also develop different strategies to target host epigenetic machinery to complete the life cycles. For instance, Respiratory syncytial virus infection elevates histone demethylase KDM5B to impair IFN-I production [7]. Epstein-Barr virus (EBV) infection not only downregulates the level of Tet methylcytosine dioxygenase 2 (TET2) but also upregulates DNMT1 level to maintain speci c DNA methylation pro le [8][9][10][11]. Additionally, HIV infection increases DNMT3b expression and FOXP3 promoter methylation, thus in uences the function of regulatory T cells [12]. These evidences highlight that epigenetic machinery is deeply involved in the pathological processes after virus infection.
DNA methylation plays important roles in regulating gene expression through DNA methyltransferases (DNMTs). DNMTs silences gene expression through making CpG dinucleotides methylated [13][14][15]. There are three members of vertebrate DNMTs: DNMT1, DNMT3a and DNMT3b. The substrate of DNMT3a and DNMT3b are hemimethylated and unmethylated DNA, while DNMT1 prefers hemimethylated DNA and thus maintains the patterns of DNA methylation during the process of DNA replication [16,17]. It is reported that after hepatitis B virus (HBV) infection, host DNMTs is regulated by viral X protein to establish aberrant epigenetic modi cations [18,19].
In the present study, we report VSV infection leads to DNMT1 protein accumulation in macrophage cell. Then, we examined VSV replication after DNMT1 functional blocking and gene silencing. Furthermore, in the context of DNMT1 inhibition, interferon responses, IRF3 expression, Irf3 promoter DNA methylation and endogenous retrovirus (ERV) transcripts expression were studied to explore the underlying mechanisms.
qRT-PCR Cells were collected after washing with PBS and RNA from the cells was extracted by using RNAiso Plus reagent. Then cDNA synthesis was done by using Reverse Transcriptase M-MLV. Real-time PCR assay was then performed to determine the transcriptional level of the genes. Gapdh gene was used as the reference control for immune genes and Hprt gene was used as the reference control for ERV genes. The data was normalized to Gapdh and Hprt gene level separately. Method of 2 -Ct has been used to present relative expression of each gene. (Table has listed all of the primers used in this paper).
Cell viability assay (CCK8 assay) Murine Raw264.7 cells were seeded into 96-well plates before treatment. Cells were treated with Thioguanine (1µM, 2.5µM, 5µM, 10µM) or DMSO for 6 and 12h. Metabolic activity of the cells was treated with CCK8 reagent 20min before testing, cell viability was measured and quanti ed by using TECAN plate spectrophotometer at 450 nm absorbance.

Statistical analysis
Unpaired Student's t-test as well as one-way ANOVA Tukey test were performed for the statistical analysis. * P < 0.05 was considered as signi cant (* P<0.05, ** P<0.01, *** P<0.001). Error bars indicate standard error.

DNMT1 inhibition impairs VSV replication
In the present work, we have found that host DNMT1 protein level in murine macrophage cell RAW264.7 has accumulated shortly (6h and 12h) after VSV (MOI = 1) infection, which is shown by Western Blotting assay (Fig.1a). In order to explore if host DNMT1 is required in the process of virus replication, we pretreated Raw264.7 cell with DNMT1 speci c inhibitor Thioguanine (1µM, 2.5µM, 5µM, 10µM) for 3h, which was followed by VSV-eGFP infection (MOI=1) for 12h. After VSV-eGFP infection, the GFP uorescence of the cells in each well have been detected by Tecan absorbance reader. The result shows that Thioguanine treatment is able to signi cantly impair EGFP signal, which demonstrates VSV propagation has been impaired signi cantly by DNMT1 inhibition (Fig. 1b). Additionally, Thioguanine effect on VSV-eGFP propagation has also been displayed in uorescence microscope observation (Fig. 1c). Furthermore, we treated macrophage cells with Thioguanine (1µM, 2.5µM, 5µM and 10µM) at different time points (6h and 12h), which was followed by CCK8 cell viability assay. We have found Thioguanine treatment could not affect cell viability at both time points, indicating the Thioguanine is not toxic to the cells (Fig. 1d). Moreover, in order to test if DNMT1 inhibition also affects VSV viral titer, we pre-treated the cells with Thioguanine 3h before VSV infection. 12h after VSV infection, cells were collected and VSV-G protein level was determined by performing Western Blotting assay. The results show that Thioguanine treatment signi cantly inhibits VSV replication (Fig. 1e). The above results demonstrate that DNMT1 pharmacological inactivation could impair VSV replication in host cells.

Dnmt1 gene silencing impairs VSV replication
To further con rm the role of DNMT1 during VSV replication, we also established a Dnmt1 gene silencing macrophage cell line through expressing Dnmt1 gene targeting shRNA, which is delivered by Lentivirus. Based on the examination of Western Blotting assay, and found DNMT1 protein has been largely reduced (Fig. 2a). The above assays demonstrate DNMT1 in macrophage cell has been successfully silenced. Then Dnmt1 silencing cell line and a control cell line were infected with VSV (MOI=1). 12h after virus infection, cells were collected for the determination of VSV-G protein level. Western Blotting assay shows VSV-G protein level has been largely reduced in Dnmt1 silencing group compared with control group (Fig.  2b). The above results demonstrate that Dnmt1 silencing could also impair VSV replication in murine macrophage cells, suggesting a supportive role of DNMT1 during VSV replication.

Loss of DNMT1 causes interferon responses
To explore the mechanism of DNMT1 loss-caused VSV suppression, we examined the level of IFNstimulated genes (ISGs) after DNMT1 inhibition and Dnmt1 gene silencing. We have found in qRT-PCR assay that Thioguanine treatment (10h) signi cantly upregulated the expression of a series of ISGs genes (Fig. 3a). This is also the case when Dnmt1 gene was silenced, as Dnmt1 knocking down induced the upregulation of a panel of ISGs (Fig. 3b). Furthermore, as ISGs expression is believed to be triggered by secreted IFNβ-activated JAK/STAT signaling, we tested Ifnb1 expression by performing qRT-PCR assay and have found that Ifnb1 mRNA level was highly enhanced after Thioguanine short term treatment (10h) or Dnmt1 gene silencing ( Fig. 3c and d). Moreover, we have also observed that IFNβ antibody blocking could fully reverse Thioguanine-induced ISG response, indicated by the transcriptional change of Mx1 and I t3 gene ( Fig. 3e and f). What is more, the observation that JAK/STAT inhibitor ruxolitinib fully reversed Thioguanine-induced ISG response further con rmed Thioguanine short-term treatment actually activates type I interferon signaling (Fig. 3g and h). The above results demonstrate that Thioguanineinduced ISGs upregulation is mainly mediated through IFNβ secretion and subsequent JAK/STAT activation.
IRF3 plays a critical role to suppress VSV in the context of DNMT1 loss IRF3 is required in the production of IFNβ. In order to uncover DNMT1 loss-induced Ifnb1 and ISGs upregulation, we detected Irf3 level after DNMT1 inhibition, and found Irf3 level was signi cantly upregulated by Thioguanine treatment (10h) (Fig. 4a), and this is also the case when Dnmt1 was knocking down (Fig. 4b). The nding suggests Thioguanine-induced IRF3 may be involved in the above ISGs up-regulation and VSV repression. To con rm this, we silenced Irf3 gene in the cells and have found Irf3 gene silencing restored Thioguanine-impaired VSV-G level (Fig. 4c). At the same time, Irf3 knocking down could also reverse Dnmt1 silencing-impaired VSV-G level (Fig. 4d). Furthermore, based on CGIs searching through MethPrimer software, CGIs were rich in Irf3 promoter region (-448 to -295 and -197 to -11). To explore if Irf3 upregulation is due its promoter CGI demethylation, we performed Methylation-Speci c PCR (MSP) assay (Fig. 4e). We have found the CGI methylation modi cation (-197 to -11 region) of Irf3 promoter has been largely reduced after Thioguanine treatment (Fig. 4f). The above results demonstrate that IRF3 plays a critical role in VSV suppression in the context of DNMT1 loss, which could be explained by the CGI demethylation in Irf3 promoter.
Thioguanine short-term treatment induces interferon response through dsRNA sensor TLR3 but does not increase endogenous retrovirus (ERV) transcripts It has been reported that long-term inhibition (Aza, 7-10 days) of DNMTs upregulates a series of endogenous retrovirus (ERV) transcripts, which interact with and activate TLR3/dsRNA complex, further upregulates ISGs in human cancer cells [20,21]. In the context of thioguanine short-term treatment (10h), we tested the level of murine ERV transcripts, but found ERV level was not signi cantly affected, indicating Thioguanine-induced ISGs upregulation is not due to the increase of ERV transcripts (Fig. 5a). However, Real-time PCR assay displays relative early ampli cation (CT value between 20-22) of murine ERV transcripts, suggesting the basal level of ERV may play a role (Fig. 5b). To con rm this, we blocked dsRNA sensor by treating the cells with TLR3/dsRNA complex inhibitor, which was followed by Thioguanine treatment. It shows that TLR3/dsRNA inhibitor is able to fully reverse Thioguanine-induced ISG (Mx1 and I t3) upregulation ( Fig. 5c and d). Moreover, we nd TLR3/dsRNA inhibitor could also suppress the basal level of ISG (Mx1 and I t3) expression without Thioguanine induction ( Fig. 5c and d). These above results suggest the basal level of ERV transcripts is required for Thioguanine-induced ISGs upregulation.

Discussion
Virus infection has been reported to regulate the expression of different epigenetic modi ers to establish persistent infection [7,8,22], which is consistent with our ndings that DNMT1 protein is elevated shortly after VSV infection. So, it is reasonable to speculate DNMT1 is involved in the process of VSV replication, and this is veri ed by thioguanine-suppressed VSV replication. Interestingly, thioguanine not only blocks DNMT1 function, but also induces degradation of DNMT1 protein [23,24]. Additionally, DNMTs inhibitors has been approved to treat cancers, causing cell apoptosis [25]. Based on the above characteristic, cell viability assay was performed and we con rmed thioguanine has no toxicity to the cells within 12h treatment, excluding the possibility that the effect is due to the impaired cell viability. Furthermore, Dnmt1 silencing also suppressed VSV, which exclude the possibility that the anti-viral effect is a pharmacological off-target effects, as off-targets may produce undesirable non-speci c cross-talks and effects [26].
Loss of DNMT1 induces ISGs upregulation. However, their promoters are rarely methylated, as classical CGIs were usually absent in their promoters [27]. Transcriptome analysis explains the absence of CGIs in ISG promoters is due to the requirement of nucleosome occupancy [28]. Actually, ISGs are directly regulated by IFN-stimulated gene factor 3 (ISGF3), which recognizes and binds to IFN-stimulated response elements (ISREs) in gene promoters [29][30][31]. ISGF3 is composed of signal transductor and activator of transcription 1 (STAT1), STAT2 and IFN-regulatory factor 9 (IRF9), triggered by IFNβ [32,33]. Indeed, we found IFNβ blocking and JAK/STAT inhibition fully reversed thioguanine-induced ISGs increase, which further suggests thioguanine does not affect ISGs expression directly. As expected, Ifnb1 level is also induced by DNMT1 loss, but CGIs were absent in Ifnb1 promoter [27]. Actually, Ifnb1 transcription is mainly directed by IRF-3/7 homodimers and/or heterodimers [34,35]. Then, we found Irf3 level was upregulated after DNMT1 inhibition, which is due to the CGIs demethylation of Irf3 promoter. It is reported that the increased (or overexpressed) level of IRF3 is able to enhance IRF3/IFN activity and induce downstream ISGs expression in different species, which may explain the above observation [36][37][38]. Importantly, we con rmed the critical role of IRF3 through the evidence that Irf3 silencing largely reversed DNMT1 loss-caused VSV suppression.
Double-stranded RNA (dsRNA) triggers protective interferon response through cytosolic RNA sensors [39]. And long-term inhibition of DNMTs induces interferon in human cancer cells, which is mediated through endogenous retroviruses (ERV)-derived dsRNA. Additionally, cells with higher basal level of ERV is correlated with higher ISGs expression [20]. In our present work, we have tested the level of several commonly studied ERV transcripts in Raw264.7 cell and found they all display a relative early ampli cation, suggesting the possibility of high basal activation of RNA sensors, which may be further ampli ed by enhanced IRF3 (Fig. 5b). Furthermore, to explore the involved RNA sensors, we blocked the function of TLR3 and found TLR3 inhibition fully reverses thioguanine-induced ISGs level as well as the basal level of ISGs, demonstrating other RNA sensors like RIG-I and MDA5 may not be involved. Consistent with our above observation, MDA5 has been reported to recognize long dsRNA, which is absent in uninfected host cell [1]. Interestingly, we found that thioguanine short-term treatment does not change the levels of commonly studied murine ERV transcripts. This suggests the interferon response enhancement is not due to the increase of ERV transcripts, which is different from DNMTs long-term inhibition-caused effect [20].

Conclusion
Collectively, we highlight the importance of host DNMT1 for VSV replication, and DNMT1 loss-caused VSV suppression is triggered by epigenetic elevated IRF3 and subsequent interferon responses. From this work we may speculate that the existence of host DNMT1 may gain time for the virus to replicate continuously in host cell through tightly controlling IFN activation.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.  VSV P primer design is based on the published article [2]. eMLV env, MMTV env, Emv2 and Hprt primers design are based on the published article [3].     Loss of DNMT1 increase interferon responses. a-d, qRT-PCR assay was performed after RAW264.7 cells were treated with 5µM thioguanine for 10h, the mRNA level of ISGs (a) and Ifnb1 (c) were examined. qRT-PCR assay was performed with Dnmt1 silencing cells and control cells, the mRNA level of ISGs (b) and Ifnb1 (d) were examined. Unpaired Student's t-test was performed for statistical analysis and the results were presented as means ± SEM (n=3). * P<0.05, ** P<0.01, *** P<0.001. e-f, qRT-PCR assay was performed after cells were treated with thioguanine (5µM), anti-IFNβ blocking antibody (0.1µg/ml) and thioguanine/antibody combination, the mRNA level of Mx1 (e) and I t3 (f) were examined. g-h, qRT-PCR assay was performed after cells were treated with thioguanine (5µM), ruxolitinib (2µM) and thioguanine/ ruxolitinib combination, mRNA level of Mx1 (g) and I t3(h) were examined. Data analysis was performed by one-way ANOVA Tukey test and the results were presented as means ± SEM (n=3). ** P<0.01, *** P<0.001.

Figure 4
Loss of DNMT1-increased IRF3 is critical for VSV suppression. a-b, qRT-PCR assay was performed after cells were treated with 5µM thioguanine (a) or transfected with Dnmt1 silencing shRNA (b), mRNA level of Irf3 was examined. Unpaired Student's t-test was performed for statistical analysis and the results were presented as means ± SEM (n=3). *** P<0.001. c, Irf3 silenced cells and control cells were pre-treated with 5µM thioguanine or DMSO before VSV (MOI=1) infection, Western Blotting was performed to detect VSV-G protein level. d, Irf3 silenced cells and control cells were transfected with shRNA-Dnmt1-lentivirus or shRNA-control-lentivirus before VSV (MOI=1) infection, Western Blotting assay was performed to detect VSV-G protein level. e, Schematic presentation of Irf3 promoter region and methylation-speci c PCR (MSP) primers design. TSS, transcriptional start site. CGI, CpG islands. MF2 and MR2 represent primers for methylated fragment detection and UF2 and UR2 represent primers for unmethylated fragment detection. f, Genomic DNA from cells treated with 5µM thioguanine or DMSO were extracted, then treated with Bisul te for MSP assay.

Figure 5
Thioguanine short-term treatment-induced interferon response is dependent on basal activity of TLR3, but not through ERV transcripts increase. a, qRT-PCR assay was performed after cells were treated with 5µM thioguanine for 10h. mRNA level of murine ERV transcripts were determined. b, Gene ampli cation curve. Real-time PCR assay displaying murine gene (Hprt, Syna, Synb, eMLV env, MMTV env and Emv2) ampli cation curve. c-d, qRT-PCR assay was performed after cells were treated with 5µM thioguanine, TLR/dsRNA complex inhibitor and thioguanine/inhibitor combination. mRNA level of Mx1 (c) and I t3 (d) were determined. Data analysis was performed by one-way ANOVA Tukey test and the results were presented as means ± SEM (n=3). *** P<0.001.

Supplementary Files
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