Schlafen 11 (Slfn11) is an interferon-stimulated gene that controls the synthesis of proteins by regulating tRNA abundance. Likely through this mechanism, Slfn11 has previously been shown to impair human immunodeficiency virus type 1 (HIV-1) infection and the expression of codon-biased open reading frames. Because replication of positive-sense single-stranded RNA [(ϩ)ssRNA] viruses requires the immediate translation of the incoming viral genome, whereas negative-sense singlestranded RNA [(Ϫ)ssRNA] viruses carry at infection an RNA replicase that makes multiple translation-competent copies of the incoming viral genome, we reasoned that (ϩ)ssRNA viruses will be more sensitive to the effect of Slfn11 on protein synthesis than (Ϫ)ssRNA viruses. To evaluate this hypothesis, we tested the effects of Slfn11 on the replication of a panel of ssRNA viruses in the human glioblastoma cell line A172, which naturally expresses Slfn11. Depletion of Slfn11 significantly increased the replication of (ϩ)ssRNA viruses from the Flavivirus genus, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV), but had no significant effect on the replication of the (Ϫ)ssRNA viruses vesicular stomatitis virus (VSV) (Rhabdoviridae family) and Rift Valley fever virus (RVFV) (Phenuiviridae family). Quantification of the ratio 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. Lowabundance tRNAs might promote optimal 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 that the cellular protein Schlafen 11 (Slfn11) impairs replication of flaviviruses, including West Nile virus (WNV), dengue virus (DENV), and Zika virus (ZIKV). However, replication of single-stranded negative 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. KEYWORDS Schlafen 11, virus restriction factors, flavivirusS uccessful viral replication depends on the ability of the virus to appropriate the host translational machinery. The innate immune response exploits this dependency to control viral replication. Many interferon (IFN)-stimulated genes (ISGs) that regulate protein translation are well known to restrict virus replication, including protein kinase R, the interferon-induced proteins with tetratricopeptide repeats family of proteins, zinc finger antiviral protein, and the 2=,5=-oligoadenylate/RNase L pathway. The Schlafen (Slfn) proteins, another family of ISGs, were first identified as being important regulators of T cell differentiation and gr...

21Schlafen 11 (Slfn11) is a ubiquitously expressed interferon stimulating gene (ISG) that 22 controls synthesis of proteins by regulating tRNA abundance. Likely through this 23 mechanism, Slfn11 has previously been shown to impair human immunodeficiency virus 24 1 (HIV-1) infection and the expression of codon-biased open reading frames. Because 25 replication of positive-sense single-stranded RNA [(+)ssRNA viruses] requires the 26 immediate translation of the incoming viral genome whereas negative sense, single 27 stranded [(-)ssRNA] viruses carry at infection an RNA replicase that makes multiple 28 translation competent copies of the incoming viral genome, we reasoned that (+)ssRNA 29 viruses will be more sensitive to the effect of Slfn11 on protein synthesis than (-)ssRNA 30 viruses. To evaluate this hypothesis, we tested the effects of Slfn11 on the replication of 31 a panel of ssRNA viruses in the human glioblastoma cell line A172, which naturally 32 expresses Slfn11. Depletion of Slfn11 in this cell line significantly increased the 33 replication of (+)ssRNA viruses from the Flavivirus family, including West Nile (WNV), 34 dengue (DENV), and Zika virus (ZIKV) but had no significant effect on the replication of 35 the (-)ssRNA viruses vesicular stomatitis (VSV, Rhabdoviridae family) and Rift Valley 36 fever (RVFV, Phenuiviridae family). Despite that WNV titers in Slfn11-deficient cells were 37 almost 100-fold higher than in cells expressing this protein; they produced approximately 38 two-fold less viral particles, as determined by PCR-based quantification of virion-39 associated WNV RNA in the cell culture supernatant. These data indicated that Slfn11 40 impairs WNV fitness but does not affect other steps of the viral life cycle including entry, 41 viral RNA replication and translation, and budding. Similarly to the proposed anti-HIV-1 42 mechanism of Slfn11, this protein prevented WNV-induced down-regulation of a subset 43 of tRNAs implicated in the translation of 19% of the viral polyprotein. Importantly, we 44 provided evidence suggesting that the broad anti-viral activity of Slfn11 requires other 3 45 cellular proteins, since overexpression of Slfn11 in cells that naturally lack the 46 expression of this protein, did not impair WNV or HIV-1 infection. In summary, this study 47 4 49 AUTHOR SUMMARY 50The host targets mechanisms that viruses have evolved to optimize replication. We 51 provide evidence that the cellular protein Schlafen 11 (Slf11) impairs replication of 52 flaviviruses, including West Nile (WNV), dengue (DENV), and Zika virus (ZIKV). 53However, replication of single-stranded, negative RNA viruses was not affected. 54Specifically, Slf11 decreases the fitness of WNV potentially by preventing virus-induced 55 modifications of the host tRNA repertoire that could lead to enhanced viral protein 56 folding. Furthermore, we demonstrated that Slf11 is not the limiting factor of this novel 57 broad anti-viral pathway. 5 59 6 84HIV-1 activity of Slf13 is specific since this protein did not affect re...

We have recently discovered a major role of the type I interferon (IFN‐I)‐induced protein Schlafen (SLFN) 11 in the control of flavivirus infection and verified its effects on HIV‐1. This protein is also relevant in controlling chemosensitivity of cancer in patients, and animal and cellular models. Therefore, understanding the mechanisms regulating SLFN11 expression during infection and chemotherapy is relevant. We have found that infection of human cells by the flavivirus West Nile virus (WNV) or HIV‐1 upregulated the expression of SLFN11. Characterization of the pathways implicated that while WNV increases SLFN11 expression via IFN‐I signaling, HIV‐1 uses an IFN‐I‐independent pathway. To further map the latter signaling pathway, we evaluated the ability of a panel of inflammatory stimuli to induce SLFN11 in primary CD4+ T cells isolated from two healthy donors. Interestingly, phorbol myristate acetate (PMA), ionomycin, and phytohemagglutinin (PHA) potently induced this protein in both donors. These compounds also upregulated SLFN11 expression in cell lines of different histological origin. However, both IFN‐I‐dependent and ‐independent pathways failed to stimulate expression of a 1.3 kb promoter cloned in a gene reporter system, suggesting that this promoter lacks cis‐acting regulatory elements that are relevant in the regulation of SLFN11 expression in cells. In summary, our data indicate that SLFN11 expression is controlled via IFN‐I‐dependent and ‐ independent pathways. Support or Funding Information Research Initiative for Scientific Enhancement funded by a grant (#R25GM069621‐11) from the National Institute of General SciencesUniversity of Texas at El Paso