The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide 1 . No approved antivirals are available to date, and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates 2 . Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available 3 . Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies 4 . A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy 5,6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects 7 . By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in hostdependency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.CRISPR is revolutionizing the use of genetic screens because the ability to completely knockout genes substantially increases the robustness of the phenotypes 5,6 . We compared the CRISPR approach in the hepatocyte cell line Huh7.5.1 with an alternative method to generate knockout alleles on a genome-wide scale: insertional mutagenesis in human haploid cells (HAP1) 8,9 (Fig. 1a). Both methods generate libraries of cells with knockout mutations in all non-essential genes. To comprehensively identify cellular genes with crucial roles in the Flaviviridae life cycles, we first infected pools of mutagenized cells with DENV serotype 2 (DENV-2). The two types of genetic screening methods showed a strong concordance in the genes enriched in the DENV-2-resistant population. Many could be functionally classified into three distinct categories, each important for proper expression of ER-targeted glycoproteins (Fig. 1b, c, Supplementary Tables 1, 2). The translocon associated protein (TRAP) complex (containing subunits SSR1, SSR2 ...

Flaviviruses including dengue virus (DENV) and Zika virus (ZIKV) cause significant human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum (ER) is a shared replication strategy, despite different clinical outcomes. While the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Due to its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, while viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Since viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. In addition, this review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.

ciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. To study the biogenesis of ciRS-7, we developed an algorithm to define its promoter and predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generates a new model for regulation of this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChIP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reverted NS1-mediated inhibition of host gene expression. A counter-screen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus. The mechanism of action was through activation of REDD1 expression and concomitant inhibition of mTORC1 via TSC1/TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1−/− cells. Viruses inhibited REDD1 expression, resulting in activation of the mTORC1 pathway. REDD1−/− cells prematurely up-regulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high levels of REDD1 down-regulated viral protein levels. Thus, REDD1 is a novel host defense factor and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.