An Integrated Transcriptomic and Meta-Analysis of Hepatoma Cells Reveals Factors That Influence Susceptibility to HCV Infection

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies

Role of Annexin A2 in the Production of Infectious Hepatitis C Virus Particles▿

Hepatitis C virus (HCV) is an important human pathogen affecting 170 million chronically infected individuals. In search for cellular proteins involved in HCV replication, we have developed a purification strategy for viral replication complexes and identified annexin A2 (ANXA2) as an associated host factor. ANXA2 colocalized with viral nonstructural proteins in cells harboring genotype 1 or 2 replicons as well as in infected cells. In contrast, we found no obvious colocalization of ANXA2 with replication sites of other positive-strand RNA viruses. The silencing of ANXA2 expression showed no effect on viral RNA replication but resulted in a significant reduction of extra- and intracellular virus titers. Therefore, it seems likely that ANXA2 plays a role in HCV assembly rather than in genome replication or virion release. Colocalization studies with individually expressed HCV nonstructural proteins indicated that NS5A specifically recruits ANXA2, probably by an indirect mechanism. By the deletion of individual NS5A subdomains, we identified domain III (DIII) as being responsible for ANXA2 recruitment. These data identify ANXA2 as a novel host factor contributing, with NS5A, to the formation of infectious HCV particles

Recruitment and activation of a lipid kinase by NS5A of the hepatitis C virus is essential for integrity of the membranous replication compartment

Hepatitis C virus (HCV) is the causative agent of chronic liver disease affecting 170 million individuals. To gain insight into host factor requirements of this important human pathogen we performed an siRNA screen of the human kinome. We identified 13 different kinases involved in HCV replication, including phosphatidyl-inositol 4 kinase III alpha (PI4KIIIα). Enzymatic activity of PI4KIIIα was critical for viral replication consistent with elevated levels of phosphatidylinositole-4-phosphate (PI4P) detected in cultured hepatocytes containing replicating virus and in liver tissue of chronic hepatitis C patients. Nonstructural protein 5A (NS5A) interacts with PI4KIIIα and stimulates kinase activity. Functional role of PI4P for integrity of the membranous HCV replication complex is indicated by a dramatic change of its ultrastructural morphology in the absence of PI4KIIIα activity. Our analysis provides evidence for direct activation of a lipid kinase by a viral protein and its link to integrity of the membranous replication complex