-Many aspects of the hepatitis C virus (HCV) life cycle have not been reproduced in cell culture, which has slowed research progress on this important human pathogen. Here, we describe a full-length HCV genome that replicates and produces virus particles that are infectious in cell culture (HCVcc). Replication of HCVcc was robust, producing nearly 10 5 infectious units per milliliter within 48 hours. Virus particles were filterable and neutralized with a monoclonal antibody against the viral glycoprotein E2. Viral entry was dependent on cellular expression of a putative HCV receptor, CD81. HCVcc replication was inhibited by interferon-a and by several HCV-specific antiviral compounds, suggesting that this in vitro system will aid in the search for improved antivirals.

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

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Since the discovery of the hepatitis C virus over 15 years ago, scientists have raced to develop diagnostics, study the virus and find new therapies. Yet virtually every attempt to dissect this pathogen has met with roadblocks that impeded progress. Its replication was restricted to humans or experimentally infected chimpanzees, and efficient growth of the virus in cell culture failed until very recently. Nevertheless hard-fought progress has been made and the first wave of antiviral drugs is entering clinical trials.virus life cycle: first, as a messenger RNA (mRNA) for translation of the viral proteins; second as a template for RNA replication; and third, as a nascent genome packaged within new virus particles. Virions presumably form by budding into the endoplasmic reticulum (ER) and leave the cell through the secretory pathway.Researchers have followed each aspect of the virus life cycle in turn. Just as infection starts from an HCV genome entering the cytoplasm and progressing through translation, replication and particle production, our understanding has progressed from having a genome sequence to understanding translation and the viral gene products, characterizing RNA replication, and establishing systems to characterize virus particles and infectivity. In this review, we summarize the current understanding of HCV replication with special emphasis on recent developments. As space is limited, we cannot be comprehensive; readers may wish to consult other reviews for detail and breadth 5,13,14. Initial studies: HCV translation and polyprotein processingThe identification of HCV yielded a partial viral genome sequence 5 . Research in the early 1990s focused on dissecting HCV gene expression and characterizing the gene products. Much has been learned about the biochemistry of three key enzymes, and structural information is now available at the atomic level for roughly half of the proteincoding region.Translation of the HCV genome, which lacks a 5፱ cap, depends on an internal ribosome entry site (IRES) within the 5፱-noncoding region (NCR). The HCV IRES binds 40S ribosomal subunits directly and avidly, bypassing the need for pre-initiation factors, and inducing an mRNA-bound conformation in the 40S subunit 15 . The IRES-40S complex then recruits eukaryotic initiation factor (eIF) 3 and the ternary complex of Met-tRNA-eIF2-GTP to form a non-canonical 48S intermediate, before a kinetically slow transition to the translationally active 80S complex 16,17 .Once initiated, translation of the HCV genome produces a large polyprotein that is proteolytically cleaved to produce 10 viral proteins (Fig. 2a). The amino-terminal one-third of the polyprotein encodes the virion structural proteins: the highly basic core (C) protein, and glycoproteins E1 and E2. After the structural region comes a small integral membrane protein, p7, which seems to function as an ion channel 18,19 . The remainder of the genome encodes the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which coordinate the intracellular process...

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here, we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, MERS-CoV, bat coronavirus HKU5 expressing the SARS-CoV-1 spike, and VSV expressing the SARS-CoV-2 spike. We identify known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways including HMGB1 and the SWI/SNF chromatin remodeling complex that are SARS-lineage and pan-coronavirus specific, respectively. We show HMGB1 regulates ACE2 expression and is critical for viral entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. Together this identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS-lineage specific and pan-coronavirus host factors that regulate susceptibility to highly pathogenic coronaviruses.

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.antivirals ͉ miR-122 ͉ RNAi ͉ HCVcc-siRNA H epatitis C virus (HCV) has a notable ability to establish persistent infections in Ϸ70% of cases, resulting in 130 million chronically infected people throughout the world (1). This prevalence has spurred considerable interest in the study of HCV-host interactions, on both cellular and molecular levels. The inability to grow HCV in cell culture led some groups to focus on the identification of cellular proteins that interact with individual HCV proteins or RNA elements, resulting in the accumulation of a large number of putative HCV-host interactions. Unfortunately, the significance of most of these with respect to the HCV life cycle is currently unknown (reviewed in ref.2). Over the past 6 years, cell culture systems have been developed that enable the characterization of HCV replication and entry (3-6). This effort recently culminated in the development of cell culture systems that reproduce the entire viral life cycle (7-9). A number of virus-host interactions have been characterized by using these experimental systems. For example, CD81 has been demonstrated to play a role in HCV entry (10-12).Sequence-specific gene silencing of RNAi is ideal for assessing the genetic phenotypes associated with virus-host interactions. We have previously shown that siRNAs are highly effective at silencing either host or viral RNAs in cells that contain replicating HCV, demonstrating th...

Summary Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP-2) recognize viral RNAs as pathogen-associated molecular patterns (PAMPs) and initiate an antiviral immune response. To understand the molecular basis of this process, we determined the crystal structure of RIG-I in complex with double-stranded RNA. The dsRNA is sheathed within a network of protein domains that include a conserved “helicase” domain (regions HEL1 and HEL2), a specialized insertion domain (HEL2i), and a C-terminal regulatory domain (CTD). A V-shaped pincer connects HEL2 and the CTD by gripping an α-helical shaft that extends from HEL1. In this way, the pincer coordinates functions of all the domains and couples RNA binding with ATP hydrolysis. RIG-I falls within the Dicer-RIG-I clade of super family 2 of helicases and this structure reveals complex interplay between motor domains, accessory mechanical domains and RNA that has implications for understanding the nanomechanical function this protein family and other ATPases more broadly.

SUMMARY Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.