Abstract
Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.
Highlights
The hepatitis C virus (HCV) is a major causative agent of acute and chronic liver diseases worldwide [1]
One of the most fascinating insights gained with these systems is the finding that infectious HCV particles assemble in close association with an intracellular lipid storage compartment termed lipid droplets
In this study we show that nonstructural protein 5A (NS5A), a component of the viral RNA replication machinery is a key factor for the formation of infectious HCV particles
Summary
The hepatitis C virus (HCV) is a major causative agent of acute and chronic liver diseases worldwide [1]. Primary infection is often asymptomatic or associated with mild and nonspecific symptoms, persistently infected persons have a high risk to develop chronic liver diseases in the course of one or several decades, the most serious outcomes being liver cirrhosis and hepatocellular carcinoma. It is this property of HCV infection and its high prevalence that explain the high medical relevance of this pathogen. The genome of HCV is a single strand RNA of positive polarity [6] This RNA has a length of about 9,600 nucleotides and a very simple organization with only one long open reading frame. It is flanked by non-translated regions at the 59 and 39 end of the genome that are required for RNA translation and replication
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