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 pre-treated Raw264.7 cell with DNMT1 specific 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 fluorescence of the cells in each well have been detected by Tecan absorbance reader. The result shows that Thioguanine treatment is able to significantly impair EGFP signal, which demonstrates VSV propagation has been impaired significantly by DNMT1 inhibition (Fig. 1b). Additionally, Thioguanine effect on VSV-eGFP propagation has also been displayed in fluorescence 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 significantly 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 confirm 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 IFN-stimulated genes (ISGs) after DNMT1 inhibition and Dnmt1 gene silencing. We have found in qRT-PCR assay that Thioguanine treatment (10h) significantly 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 Ifit3 gene (Fig. 3e and f). What is more, the observation that JAK/STAT inhibitor ruxolitinib fully reversed Thioguanine-induced ISG response further confirmed Thioguanine short-term treatment actually activates type I interferon signaling (Fig. 3g and h). The above results demonstrate that Thioguanine-induced 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 significantly upregulated by Thioguanine treatment (10h) (Fig. 4a), and this is also the case when Dnmt1 was knocking down (Fig. 4b). The finding suggests Thioguanine-induced IRF3 may be involved in the above ISGs up-regulation and VSV repression. To confirm 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-Specific PCR (MSP) assay (Fig. 4e). We have found the CGI methylation modification (-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 significantly 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 amplification (CT value between 20-22) of murine ERV transcripts, suggesting the basal level of ERV may play a role (Fig. 5b). To confirm 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 Ifit3) upregulation (Fig. 5c and d). Moreover, we find TLR3/dsRNA inhibitor could also suppress the basal level of ISG (Mx1 and Ifit3) 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.