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...

Vesicular stomatitis virus (VSV) emerges periodically from its focus of endemic transmission in southern Mexico to cause epizootics in livestock in the US. The ecology of VSV involves a diverse, but largely undefined, repertoire of potential reservoir hosts and invertebrate vectors. As part of a larger program to decipher VSV transmission, we conducted a study of the spatiotemporal dynamics of Simulium black flies, a known vector of VSV, along the Rio Grande in southern New Mexico, USA from March to December 2020. Serendipitously, the index case of VSV-Indiana (VSIV) in the USA in 2020 occurred at a central point of our study. Black flies appeared soon after the release of the Rio Grande’s water from an upstream dam in March 2020. Two-month and one-year lagged precipitation, maximum temperature, and vegetation greenness, measured as Normalized Difference Vegetation Index (NDVI), were associated with increased black fly abundance. We detected VSIV RNA in 11 pools comprising five black fly species using rRT-PCR; five pools yielded a VSIV sequence. To our knowledge, this is the first detection of VSV in the western US from vectors that were not collected on premises with infected domestic animals.

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...

HIV-1 maturation can be impaired by altering protease (PR) activity, the structure of the Gag-Pol substrate, or the molecular interactions of viral structural proteins. Here we report the synthesis and characterization of new cationic N,N-dimethyl[70]fulleropyrrolidinium iodide derivatives that inhibit more than 99% of HIV-1 infectivity at low micromolar concentrations. Analysis of the HIV-1 life cycle indicated that these compounds inhibit viral maturation by impairing Gag and Gag-Pol processing. Importantly, fullerene derivatives 2a-c did not inhibit in vitro PR activity and strongly interacted with HIV immature capsid protein in pull-down experiments. Furthermore, these compounds potently blocked infectivity of viruses harboring mutant PR that are resistant to multiple PR inhibitors or mutant Gag proteins that confer resistance to the maturation inhibitor Bevirimat. Collectively, our studies indicate fullerene derivatives 2a-c as potent and novel HIV-1 maturation inhibitors.