Hepatitis C virus, a major human pathogen, produces infectious virus particles with several unique features, such as an ability to interact with serum lipoproteins, a dizzyingly complicated process ...of virus entry, and a pathway of virus assembly and release that is closely linked to lipoprotein secretion. Here, we review these unique features, with an emphasis on recent discoveries concerning virus particle structure, virus entry and virus particle assembly and release.
Intracellular RIG-I-like receptors (RLRs, including RIG-I, MDA-5, and LGP2) 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 (dsRNA). 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 the superfamily 2 helicases, and this structure reveals complex interplay between motor domains, accessory mechanical domains, and RNA that has implications for understanding the nanomechanical function of this protein family and other ATPases more broadly.
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► Structural analysis of RIG-I shows how it recognizes and encases double-stranded RNA ► A pincer motif formed by the helicase domain organizes RIG-I for RNA binding ► Functional analyses confirm the use of the pincer motif in vitro and in vivo ► Structure offers mechanistic insight into SF2 family and Dicer-related helicases
The innate immune receptor RIG-I wraps around viral dsRNA. Movements within the helicase domain suggest a mechanism for complex assembly and oligomerization of RIG-I along the RNA.
•New direct-acting antivirals (DAAs) eliminate persistent hepatitis C virus infection in >95% of patients.•DAAs are so successful, some question the need for continued research.•Critical public ...health, translational and clinical challenges remain.•A vaccine that prevents chronic infection is essential for global HCV eradication.•A unique research “toolbox” offers opportunities that warrant sustained funding.
The development and clinical implementation of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C. Infection with any hepatitis C virus (HCV) genotype can now be eliminated in more than 95% of patients with short courses of all-oral, well-tolerated drugs, even in those with advanced liver disease and liver transplant recipients. DAAs have proven so successful that some now consider HCV amenable to eradication, and continued research on the virus of little remaining medical relevance. However, given 400,000 HCV-related deaths annually important challenges remain, including identifying those who are infected, providing access to treatment and reducing its costs. Moreover, HCV infection rarely induces sterilizing immunity, and those who have been cured with DAAs remain at risk for reinfection. Thus, it is very unlikely that global eradication and elimination of the cancer risk associated with HCV infection can be achieved without a vaccine, yet research in that direction receives little attention. Further, over the past two decades HCV research has spearheaded numerous fundamental discoveries in the fields of molecular and cell biology, immunology and microbiology. It will continue to do so, given the unique opportunities afforded by the reagents and knowledge base that have been generated in the development and clinical application of DAAs. Considering these critical challenges and new opportunities, we conclude that funding for HCV research must be sustained.
The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including ...single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.
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•Derivation of human neocortical and spinal cord neuroepithelial stem (NES) cells•Zika virus (ZIKV) infects NES cells and radial glia, impairing mitosis and survival•ZIKV induces mitochondrial sequestration of centrosomal phospho-TBK1•Nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death
Onorati et al. establish neuroepithelial stem (NES) cells as a model for studying human neurodevelopment and ZIKV-induced microcephaly. Together with analyses in human brain slices and microcephalic human fetal tissue, they find that ZIKV predominantly infects NES and radial glial cells, reveal a pivotal role for pTBK1, and find that nucleoside analogs inhibit ZIKV replication, protecting NES cells from cell death.
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 (IFNAR) 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.
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•Zika virus replicates in the vaginal tract of wild-type virgin and pregnant mice•Innate RNA sensors and type I interferons control vaginal Zika virus replication•Vaginal Zika virus infection in early pregnancy leads to fetal growth restriction•Vaginal Zika virus infection of pregnant dams leads to fetal brain infection
Vaginal mucosa is permissive to the replication of Zika virus, and infection through this route can lead to fetal brain infection even in mice with an intact immune system.
Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA ...viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.
RNA is a versatile macromolecule that accommodates functional information in primary sequence and secondary and tertiary structure. We use a combination of chemical probing, RNA structure modeling, ...comparative sequence analysis, and functional assays to examine the role of RNA structure in the hepatitis C virus (HCV) genome. We describe a set of conserved but functionally diverse structural RNA motifs that occur in multiple coding regions of the HCV genome, and we demonstrate that conformational changes in these motifs influence specific stages in the virus’ life cycle. Our study shows that these types of structures can pervade a genome, where they play specific mechanistic and regulatory roles, constituting a “code within the code” for controlling biological processes.
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•The genome of HCV is folded into specific RNA structures•HCV genomic structures are conserved across multiple genotypes•Elaborate RNA structures are present within protein-coding sequences•Genetic manipulation of these structures affects replication and infectivity
Using biochemistry and viral genetics, the RNA genome of HCV was shown to contain numerous well-conserved RNA structures within protein-coding regions. Genetic manipulation of these structures alters the ability of HCV to replicate and infect, thereby demonstrating that RNA sequences can store multiple layers of information.
Hepatitis C virus (HCV) core protein is directed to the surface of lipid droplets (LD), a step that is essential for infectious virus production. However, the process by which core is recruited from ...LD into nascent virus particles is not well understood. To investigate the kinetics of core trafficking, we developed methods to image functional core protein in live, virus-producing cells. During the peak of virus assembly, core formed polarized caps on large, immotile LDs, adjacent to putative sites of assembly. In addition, LD-independent, motile puncta of core were found to traffic along microtubules. Importantly, core was recruited from LDs into these puncta, and interaction between the viral NS2 and NS3-4A proteins was essential for this recruitment process. These data reveal new aspects of core trafficking and identify a novel role for viral nonstructural proteins in virus particle assembly.
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.
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