Cellular and molecular biology of HCV infection and hepatitis

bHepatitis C virus (HCV) infection is believed to begin with interactions between cell-free HCV and cell receptors that include CD81, scavenger receptor B1 (SR-B1), claudin-1 (CLDN1), and occludin (OCLN). In this study, we have demonstrated that HCV spreading from infected hepatocytes to uninfected hepatocytes leads to the transfer of HCV and the formation of infection foci and is cell density dependent. This cell-cell contact-mediated (CCCM) HCV transfer occurs readily and requires all these known HCV receptors and an intact actin cytoskeleton. With a fluorescently labeled replication-competent HCV system, the CCCM transfer process was further dissected by live-cell imaging into four steps: donor cell-target cell contact, formation of viral puncta-target cell conjugation, transfer of viral puncta, and posttransfer. Importantly, the CCCM HCV transfer leads to productive infection of target cells. Taken together, these results show that CCCM HCV transfer constitutes an important and effective route for HCV infection and dissemination. These findings will aid in the development of new and novel strategies for preventing and treating HCV infection.

mentioning

confidence: 99%

Cell-Cell Contact-Mediated Hepatitis C Virus (HCV) Transfer, Productive Infection, and Replication and Their Requirement for HCV Receptors

Liu

2013

“…The 5= NTR comprises an internal ribosome entry site (IRES) that directs the expression of a polyprotein precursor (4,5). The polyprotein is cleaved into structural (core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins that, in association with cellular factors, form a membrane-associated replicase complex.…”

mentioning

confidence: 99%

Low Oxygen Tension Enhances Hepatitis C Virus Replication

Vassilaki

Kalliampakou

Kotta‐Loizou

et al. 2013

; Applied Molecular Virology, Institut Pasteur Korea, Seongnam-si, South Korea g Low oxygen tension exerts a significant effect on the replication of several DNA and RNA viruses in cultured cells. In vitro propagation of hepatitis C virus (HCV) has thus far been studied under atmospheric oxygen levels despite the fact that the liver tissue microenvironment is hypoxic. In this study, we investigated the efficiency of HCV production in actively dividing or differentiating human hepatoma cells cultured under low or atmospheric oxygen tensions. By using both HCV replicons and infectionbased assays, low oxygen was found to enhance HCV RNA replication whereas virus entry and RNA translation were not affected. Hypoxia signaling pathway-focused DNA microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) analyses revealed an upregulation of genes related to hypoxic stress, glycolytic metabolism, cell growth, and proliferation when cells were kept under low (3% [vol/vol]) oxygen tension, likely reflecting cell adaptation to anaerobic conditions. Interestingly, hypoxia-mediated enhancement of HCV replication correlated directly with the increase in anaerobic glycolysis and creatine kinase B (CKB) activity that leads to elevated ATP production. Surprisingly, activation of hypoxia-inducible factor alpha (HIF-␣) was not involved in the elevation of HCV replication. Instead, a number of oncogenes known to be associated with glycolysis were upregulated and evidence that these oncogenes contribute to hypoxia-mediated enhancement of HCV replication was obtained. Finally, in liver biopsy specimens of HCV-infected patients, the levels of hypoxia and anaerobic metabolism markers correlated with HCV RNA levels. These results provide new insights into the impact of oxygen tension on the intricate HCVhost cell interaction. H epatitis C virus (HCV) infection causes a wide range of clinical manifestations, from a healthy carrier state to acute and chronic hepatitis that can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. Nearly 3% of the world's population is chronically infected with HCV (1, 2), and current therapeutic approaches are not broadly effective (3).HCV is a positive-strand RNA virus with a 9.6-kb genome that is flanked at both termini by conserved, nontranslated regions (NTRs), required for RNA translation and replication. The 5= NTR comprises an internal ribosome entry site (IRES) that directs the expression of a polyprotein precursor (4, 5). The polyprotein is cleaved into structural (core, E1, E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) proteins that, in association with cellular factors, form a membrane-associated replicase complex. This copies the viral positive-strand RNA into a negative-strand intermediate that serves as the template for the synthesis of progeny genomes. The alternative reading frame (ARFP) or coreϩ1 and minicore proteins, with as-yet-unknown functions, appear to be synthesized from the core region by alternative translation mechanisms (6, 7).Studies of the...

“…epatitis C virus (HCV) is a positive-strand RNA virus encoding a polyprotein that is cleaved into 10 proteins, including three structural proteins (core, E1, and E2), a putative ion channel (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42). Propelled by the development of both subgenomic replicons (8,31) and a cell culture-based infection system (HCVcc) (9,29,49,54), research has garnered a wealth of information on the role of each of these proteins in the life cycle of the virus.…”

mentioning

confidence: 99%

Suppression of Viral RNA Binding and the Assembly of Infectious Hepatitis C Virus Particles In Vitro by Cyclophilin Inhibitors

Nag

Robotham

Tang

2012

Self Cite

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) is an indispensable component of the HCV replication and assembly machineries. Although its precise mechanism of action is not yet clear, current evidence indicates that its structure and function are regulated by the cellular peptidylprolyl isomerase cyclophilin A (CyPA). CyPA binds to proline residues in the C-terminal half of NS5A, in a distributed fashion, and modulates the structure of the disordered domains II and III. Cyclophilin inhibitors (CPIs), including cyclosporine (CsA) and its nonimmunosuppressive derivatives, inhibit HCV infection of diverse genotypes, both in vitro and in vivo. Here we report a mechanism by which CPIs inhibit HCV infection and demonstrate that CPIs can suppress HCV assembly in addition to their well-documented inhibitory effect on RNA replication. Although the interaction between NS5A and other viral proteins is not affected by CPIs, RNA binding by NS5A in cell culture-based HCV (HCVcc)-infected cells is significantly inhibited by CPI treatment, and sensitivity of RNA binding is correlated with previously characterized CyPA dependence or CsA sensitivity of HCV mutants. Furthermore, the difference in CyPA dependence between a subgenomic and a full-length replicon of JFH-1 was due, at least in part, to an additional role that CyPA plays in HCV assembly, a conclusion that is supported by experiments with the clinical CPI alisporivir. The host-directed nature and the ability to interfere with more than one step in the HCV life cycle may result in a higher genetic barrier to resistance for this class of HCV inhibitors. Hepatitis C virus (HCV) is a positive-strand RNA virus encoding a polyprotein that is cleaved into 10 proteins, including three structural proteins (core, E1, and E2), a putative ion channel (p7), and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (42). Propelled by the development of both subgenomic replicons (8, 31) and a cell culture-based infection system (HCVcc) (9,29,49,54), research has garnered a wealth of information on the role of each of these proteins in the life cycle of the virus. The structural proteins and p7 are needed for virion assembly but not RNA replication, whereas the nonstructural proteins are probably involved in both replication and infectious virus production. In addition to the requisite protease and polymerase activities, HCV also encodes proteins that perform specialized functions, such as deforming the membranes (to generate a microenvironment for RNA replication) or scaffolding (to bring the appropriate proteins together for virion assembly) (14,24,37,41).HCV infects ϳ3% of the world's population and is a major risk factor for liver cirrhosis and hepatocellular carcinoma (5, 47). Current treatment comprises pegylated interferon (IFN), the nucleoside analog ribavirin, and recently approved direct-acting antivirals (DAAs) that inhibit the HCV NS3 protease. Despite the success of this triple therapy, drug resistance against the DAAs develops quickly (38), presuma...