[PMC free article] [PubMed] [CrossRef] [Google Scholar] 24

[PMC free article] [PubMed] [CrossRef] [Google Scholar] 24. (?)ssRNA viruses. To evaluate this hypothesis, we tested the effects of Slfn11 within the replication of a panel HCV-IN-3 of ssRNA viruses in the human being glioblastoma cell collection A172, which naturally expresses Slfn11. Depletion of Slfn11 significantly improved the replication of (+)ssRNA viruses from your genus, including Western Nile computer virus (WNV), dengue computer virus (DENV), and Zika computer virus (ZIKV), but experienced no significant effect on the replication of the (?)ssRNA viruses vesicular stomatitis computer virus (VSV) (family) and Rift Valley fever computer virus (RVFV) (family). Quantification of the percentage of genome-containing viral particles to PFU indicated that Slfn11 impairs WNV infectivity. Intriguingly, Slfn11 prevented WNV-induced downregulation of a subset of tRNAs implicated in the translation of 11.8% of the viral polyprotein. Low-abundance tRNAs might promote ideal protein folding and enhance viral infectivity, as previously reported. In summary, this study demonstrates that Slfn11 restricts flavivirus replication by impairing viral infectivity. IMPORTANCE We provide evidence the cellular protein Schlafen 11 (Slfn11) impairs replication of flaviviruses, including Western Nile computer virus (WNV), dengue computer virus (DENV), and Zika computer virus (ZIKV). However, replication of single-stranded unfavorable RNA viruses was not affected. Specifically, Slfn11 decreases the infectivity of WNV potentially by preventing virus-induced modifications of the host tRNA repertoire that could lead to enhanced viral protein folding. Furthermore, we demonstrate that Slfn11 is not the limiting factor of this novel broad antiviral pathway. family) and Rift Valley fever virus (RVFV) (family) was analyzed, highlighting the specificity of this antiviral activity. Furthermore, quantification of the ratio of genome-containing HCV-IN-3 viral particles to PFU in the supernatant of infected cells exhibited that Slfn11 impairs WNV infectivity. Analysis of the tRNA repertoire indicated that Slfn11 prevented the WNV-induced downregulation of the expression of a subset of tRNAs implicated in the translation of the viral polyprotein. Finally, we found that cells lacking endogenous Slfn11 failed to support the antiviral activity of exogenously introduced Slfn11. In summary, our data demonstrate that Slfn11 decreases viral infectivity, restricting the replication of flaviviruses. (This article was submitted to an online preprint archive [9].) RESULTS WNV contamination induces expression of Slfn11. To determine whether WNV contamination modulates levels of Slfn11 expression, we characterized cells of the human glioblastoma cell line A172. These cells were demonstrated to be highly susceptible to WNV contamination and to modulate the expression of a subset of genes implicated in neurodegeneration (10). To further verify the suitability of this cellular model for WNV replication, we compared the susceptibilities of Vero and A172 cells to WNV. Cells were infected at a multiplicity of contamination (MOI) of 1 1, and the level of WNV envelope protein (E) was determined by flow cytometry analysis with a specific antibody. Results from two impartial contamination experiments indicated that A172 cells were as susceptible as Vero cells to WNV contamination. At 12?h postinfection HCV-IN-3 (p.i.), 11% 0.22% of A172 cells and 15% 0.85% of Vero cells expressed WNV E protein, and 100% of the cells of both cell lines were positive for this structural viral protein at 32?h postinfection. A172 cells were infected with WNV at an MOI of HCV-IN-3 0.1, and viral replication and expression of Slfn11 were determined at different times p.i. by a plaque assay and immunoblotting, respectively (Fig. 1a and HSPA1 ?andb).b). Viral replication was detectable by as early as 8?h p.i. and peaked by 32?h after contamination (Fig. 1a). Corresponding with the peak of viral replication, we detected a sustained increase in the basal levels of Slfn11 after 40?h p.i. (Fig. 1b). Densitometry analysis of immunoblots from two impartial contamination experiments indicated that WNV contamination caused a 3.5-fold increase in the -tubulin-normalized Slfn11 protein levels after 40?h p.i. Therefore, these data indicated that Slfn11 is usually upregulated by WNV contamination. Open in a separate window FIG 1 Kinetics of WNV replication, type I interferon production, and Slfn11 expression in A172 cells. (a) WNV replication in A172 cells. Cells were infected with WNV at an MOI of 0.1, and viral.