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

Figures

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  Fig. 1. ZIKV replicates and IRF2 is induced in A549 cells. A549 cells were infected with ZIKV (MOI = 0.5) or left uninfected and total cellular RNAs were extracted at different hours-post-infection (hpi). RNA levels of ZIKV NS5 (a), IRF2 (b), ISG15 (c) and IFIT1 (d) were measured by RT-qPCR. Protein levels of ZIKV-NS1 and IRF2 were measured by western blots (d). Data were presented as mean ± standard deviation (SD) representative of at least three independent experiments

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  Fig. 2. Knockdown of IRF2 promoted ZIKV replication independent of type I IFN signaling. A549 cells were transfected with siIRF2 or siNC and infected with ZIKV at MOI of 0.5. After 24 h, IFNβ was added to appropriate wells to a final concentration of 100 IU/mL. Total RNAs and proteins were extracted 24 h later, and selected gene expression was tested by RT-qPCR (b, f, g) and western blot (a, c). The relative ZIKV production as affecting of siIRF2 and IFNβ were significantly different (siIRF2*IFNβ, P = 0.000, F = 919.4). The relative ISG15 production as affecting of siIRF2 and IFNβ were significantly different (siIRF2*IFNβ, P = 0.012, F = 19.1). The relative IFIT1 production as affecting of siIRF2 and IFNβ were significantly different (siIRF2*IFNβ, P = 0.000, F = 224.4). 2FTGH and U5A cells were transfected with siIRF2 or siNC and infected with ZIKV (MOI = 0.5). After 24 h, mRNA expression of ZIKV-NS5 was examined with RT-qPCR (d, e). siIRF2 or siNC were transfected into A549 cells, IFNβ, ISG15 and IFIT1 RNA levels were extracted and examined with RT-qPCR at 24 h later (h, i, j). Data was shown as mean ± standard deviation representative of at least three independent experiments (t test,…

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  Fig. 3. IRF2 silencing decreased FAM111A expression and knockdown of FAM111A or RFC3 promoted ZIKV replication in both 2FTGH and U5A cells. 2FTGH and U5A cells were transfected with siNC or siIRF2 for 24 h, and then the cells were infected with ZIKV at MOI of 0.5. Expression levels of FAM111A were examined both at mRNA (a, b) and protein (c, d) levels 48 h post transfection. 2FTGH and U5A cells were transfected with siNC, siFAM111A, or siRFC3 for 24 h, and then the cells were infected with ZIKV at MOI of 0.5. Expression levels of ZIKV-NS5 were examined both at RNA (e, f) and protein levels (g, h) 48 h post transfection. ISG15 and IFIT1 were examined at mRNA levels with RT-qPCR (i–l). The relative ZIKV production in 2FTGH cells between siNC and siFAM111A (P = 0.03, F = 15.2) or siRFC3 (P = 0.004, F = 68.9) were significantly different. The relative ZIKV production in U5A cells between siNC and siFAM111A (P = 0.002, F = 90.1) or siRFC3 (P = 0.001, F = 145.6) were significantly different. Data were presented as mean ± standard deviation representative of at least three independent experiments (t test, *P < 0.05; **P < 0.01)

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  Fig. 4. Knockdown of FAM111A or RFC3 abolished the inhibitory effect of IRF2 overexpression on ZIKV replication. The experiments were divided into 8 groups of 2FTGH cells which were transfected with pEmpty, pIRF2, siNC, siFAM111A, siRFC3, pIRF2 + siNC, pIRF2 + siFAM111A orpIRF2 + siRFC3, respectively, for 24 h. All the cells were infected with ZIKV at MOI of 0.5 and the mRNA expression levels of FAM111A (a), IRF2 (b) and ZIKV (d) were measured by RT-qPCR at 24 h-post-infection. 2FTGH cells was transfected with pEmpty or pIRF2 plasmid before being infected with ZIKV (MOI = 0.5) for 24 h. The relative ZIKV production in 2FTGH cells between pEmpty and pIRF2 (P = 0.047, F = 9.2) were significantly different. The relative ZIKV production in 2FTGH cells between siNC and siFAM111A (P = 0.000, F = 93.2) or siRFC3 (P = 0.000, F = 123.6). The relative ZIKV production in 2FTGH cells between pIRF2 and siFAM111A (pIRF2*siFAM111A, P = 0.000, F = 44.5) or siRFC3 (pIRF2*siFAM111A, P = 0.002, F = 18) were significantly different. The IRF2 protein expression (c) was detected with western blots (t test, *P < 0.05; **P < 0.01)