IRF2 inhibits ZIKV replication by promoting FAM111A expression to enhance the host restriction effect of RFC3

A549 cells (human non-small-cell lung cancer cell line) were purchased from West China Hospital of Sichuan University. Mutant U5A cells were derived from the parental human fibrosarcoma cells (2fTGH cells) and had IFNARI gene-deficient for a component of the IFNα/β receptor were kindly provided by Dr. Wenyu Lin (Harvard Medical School, USA) [25, 26]. A549 cells, 2FTGH cells and U5A cells were grown in Dulbecco′s Modified Eagle′s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and 100 μg/mL streptomycin sulfate. Cells were cultured in a 5% CO2 humidified incubator at 37 °C. ZIKV (GZ01 strain) was generously provided by professor Chengfeng Qin (Institute of Microbiology and Epidemiology, China) and was propagated in mosquito C6/36 cells as described in previous publication [27]. For virus infection, cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.5 with serum-free and antibiotic-free medium and incubate for 4 h. Cells were then washed with PBS for 3 times and re-fed with normal medium.

Total RNAs were extracted using Trizol reagent (Invitrogen, USA) according to the manufacturer′s instructions. cDNA was synthesized using the First Strand cDNA Synthesis Kit (Bio-rad, USA). Real-time PCR was performed with Fast start Universal SYBR Green Master Mix (Roche, USA). Normalization and quantitation were performed using GAPDH RNA and the ΔΔCt method. The selected mRNA real-time PCR primers were listed in Table 1.

The cells were washed 3 times with PBS, and then lysed by adding 100 μL of Radio Immunoprecipitation Assay (RIPA) (Beyotime, China) containing PMSF. Cell lysates were centrifugated and protein concentration was quantified with the Protein Assay Kit (Beyotime, China). Protein samples were separated by SDS–PAGE gels and transferred to the PVDF membranes (Millipore, USA). The membranes were blocked using 5% bovine serum albumin (BSA) and then incubated with the specific primary antibody in 4 °C for 16 hours. The primary antibodies included rabbitanti-IRF2 (Proteintech, China), mouse anti-ZIKV NS1 (GeneTex, USA), rabbitanti-RFC3 (Proteintech, China), rabbitanti-IRF2(Abcam, UK) and mouse anti-GAPDH (GeneTex_, USA). The secondary antibody used was HRP-labeled goat anti-mouse IgG or anti-rabbit (Beyotime, China). The protein bands were detected using the ECL Western Blotting Analysis System (Mllipore, USA) on Image Quant LAS 4000 mini (GE, USA).

Specific small interfering RNAs (siRNAs) were purchased from Sangon company (shanghai, China). The sense and antisense sequences of the siRNAs are listed in Table 2. pIRF2 (plasmid IRF2) and pEmpty (empty control) were purchased from vigene biosciences company (Shandong, China). Double-stranded siRNA and plasmid were transfected into cells with RNAiMax (Invitrogen, USA) according to the manufacturer′s instruction.

To investigate whether the expression levels of IRF2 were changed following ZIKV replication by time, A549 cells were infected with ZIKV at a MOI of 0.5. mRNA and protein expression levels of ZIKV and IRF2 were measured at 12, 24, 48 and 72 h-post-infection (hpi) by RT-qPCR and western blots. We calculated the relative expression of ZIKV RNA and IRF2 by normalizing ZIKV NS5 and IRF2 to GAPDH, respectively. As shown in (Fig. 1a, b, e), ZIKV can steadily replicate in A549 cells, and compared with the uninfected control, IRF2 expression is gradually increased from 12 to 72 h post-infection. In addition, to assess effect of mainly antiviral factor (ISGs) in ZIKV-infected A549 cells, we examine expression levels of ISG15 and IFIT1 at different time (Fig. 1c, d). These results indicated that ZIKV can infect and replicate steadily in A549 cells and IRF2, ISG15 and IFIT1 expression was increased upon ZIKV infection.

RNA interference was employed to dissect the role of IRF2 in ZIKV replication. We found IRF2 siRNA markedly knocked down IRF2 protein levels (Fig. 2a) and promoted ZIKV replication both at mRNA and protein levels in A549 cells (Fig. 2b, c). As IRF2 has been reported to be a negative regulator of type-I interferon pathway, we next checked mRNA expression of two classical interferon stimulated genes, ISG15 and IFIT1 in IRF2-silenced A549 cells. We found that silencing of IRF2 indeed increased the mRNA expression of ISG15 and IFIT1 significantly (Fig. 2f, g), but seems contradictory to the stimulating effect on ZIKV because these ISGs are classical anti-viral proteins [28]. To confirm the type-I interferon pathway is intact in A549 cells, we were able to detect the significant inhibitory effect of IFNβ on ZIKV replication by treating the ZIKV-infected cells with 100IU/mL IFNβ. In addition, compared to IFNβ treatment alone, combination of IFNβ treatment and IRF2 silencing increased ISG15 (Fig. 2f) and IFIT1 (Fig. 2g) mRNA expression to a higher level and inhibited ZIKV replication more significantly both at RNA (Fig. 2b) and protein levels (Fig. 2c). as the control, we examined IFNβ, ISG15 and IFIT1 RNA levels (Fig. 2h–j). All these data pointed out the type-I IFN-activated Jak/STAT signaling is intact in A549 cells. Furthermore, we also used an IFNAR-deficient U5A cells to verify whether the stimulating effect of IRF2 silencing on ZIKV replication is dependent on the type-I IFN signaling pathway. We found that IRF2 siRNA increased ZIKV replication as shown by NS5 mRNA expression in both U5A and its parental 2FTGH cells (Fig. 2d, e). Collectively, these results indicated that IRF2 regulate ZIKV replication in an IFN-independent manner.

Panda et al. [21] thought that IRF2 regulated FAM111A expression, which could form a complex with RFC to restrict poxviruses replication in human cells. In order to investigate whether IRF2 silencing promoted ZIKV replication by regulating the expression of FAM111A, we transfected IRF2 siRNA to 2FTGH and U5A cells and then infected with ZIKV at MOI = 0.5. As shown in Fig. 3a–d, IRF2 knockdown decreased the expression of FAM111A significantly. Furthermore, knockdown of FAM111A or subunit of RFC (RFC3) pronouncedly promoted ZIKV replication in both 2FTGH and U5A cells as shown in Fig. 3e–h. To assess whether I type IFN signaling pathway was activited by FAM111A and RFC3 knockdown, we examined ISG15 and IFIT1 in ZIKV-infected 2FTGH and U5A cells with siFAM111A and siRFC3. Figure 3i–l showed siFAM111A and siRFC3 didn't affect ISG15 and IFIT1 expression. These data indicated that IRF2 may modulate ZIKV replication by regulating FAM111A and RFC3 expression.

In order to further confirm the role of IRF2 in ZIKV replication through FAM111A and RFC3, we investigated the effect of IRF2 overexpression (Fig. 4b, c) on ZIKV replication in FAM111A or RFC3 knocked-down 2FTGH cells (Fig. 4d). We found overexpression of IRF2 increased FAM111A mRNA level by 2-fold (Fig. 4a) and inhibited ZIKV replication (Fig. 4d). However, this inhibitory effect of IRF2 on ZIKV replication was abolished in FAM111A or RFC3 knocked-down 2FTGH cells (Fig. 4d).