SUMMARY The RNA modification N6-methyladenosine (m6A) post-transcriptionally regulates RNA function. The cellular machinery that controls m6A includes methyltransferases and demethylases that add or remove this modification as well as m6A-binding YTHDF proteins that promote the translation or degradation of m6A-modified mRNA. We demonstrate that m6A modulates infection by hepatitis C virus (HCV). Depletion of m6A-methyltransferases or an m6A-demethylase respectively increases and decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m6A sites across the HCV genome and determine that inactivating m6A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m6A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m6A. Together, this work identifies m6A as a conserved regulatory mark across Flaviviridae genomes.

B-cell depleting therapies may lead to prolonged disease and viral shedding in individuals infected with SARS-CoV-2 and this viral persistence raises concern for viral evolution. We report on the sequencing of early and late samples from a 335-day infection in an immunocompromised patient. The virus accumulated a unique deletion in the amino-terminal domain of the spike protein, and complete deletion of ORF7b and ORF8, the first report of its kind in an immunocompromised patient. Overall, the unique viral mutations found in this study highlight the importance of analyzing viral evolution in protracted SARS-CoV-2 infection, especially in immunosuppressed hosts.

The molecular mechanisms that govern hepatitis C virus (HCV) assembly, release, and infectivity are still not yet fully understood. In the present study, we sequenced a genotype 2A strain of HCV (JFH-1) that had been cell culture adapted in Huh-7.5 cells to produce nearly 100-fold-higher viral titers than the parental strain. Sequence analysis identified nine mutations in the genome, present within both the structural and nonstructural genes. The infectious clone of this virus containing all nine culture-adapted mutations had 10-fold-higher levels of RNA replication and RNA release into the supernatant but had nearly 1,000-fold-higher viral titers, resulting in an increased specific infectivity compared to wild-type JFH-1. Two mutations, identified in the p7 polypeptide and NS5B RNA-dependent RNA polymerase, were sufficient to increase the specific infectivity of JFH-1. We found that the culture-adapted mutation in p7 promoted an increase in the size of cellular lipid droplets following transfection of viral RNA. In addition, we found that the culture-adaptive mutations in p7 and NS5B acted synergistically to enhance the specific viral infectivity of JFH-1 by decreasing the level of sphingomyelin in the virion. Overall, these results reveal a genetic interaction between p7 and NS5B that contributes to virion specific infectivity. Furthermore, our results demonstrate a novel role for the RNA-dependent RNA polymerase NS5B in HCV assembly. IMPORTANCEHepatitis C virus assembly and release depend on viral interactions with host lipid metabolic pathways. Here, we demonstrate that the viral p7 and NS5B proteins cooperate to promote virion infectivity by decreasing sphingomyelin content in the virion. Our data uncover a new role for the viral RNA-dependent RNA polymerase NS5B and p7 proteins in contributing to virion morphogenesis. Overall, these findings are significant because they reveal a genetic interaction between p7 and NS5B, as well as an interaction with sphingomyelin that regulates virion infectivity. Our data provide new strategies for targeting host lipid-virus interactions as potential targets for therapies against HCV infection. Hepatitis C virus (HCV), a positive-sense single-stranded RNA virus and a member of the Flaviviridae family, infects nearly 170 million people worldwide (1, 2). HCV infection is the leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (3). While the previous interferon-based therapies for treatment of hepatitis C were effective in only 40% of those infected with genotype 1 virus, the most prevalent HCV genotype in the United States (4), newly developed therapies for hepatitis C now use interferon-free direct-acting antiviral (DAA) regimens, with over 90% success rates (5). However, antiviral resistance is still a problem, and a vaccine is not yet available for prevention of HCV transmission.The HCV RNA genome is translated into a single polyprotein from an internal ribosome entry site located in the 5= untranslated region of the genome. The resu...