Essential Role of Domain III of Nonstructural Protein 5A for Hepatitis C Virus Infectious Particle Assembly

Top Cited Papers
Open Access
Publisher Website
Google Scholar
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. The hepatitis C virus (HCV) is a major cause of acute and chronic liver diseases worldwide. In spite of high medical need there is no selective antiviral therapy available and a prophylactic vaccine is not in sight. Their development requires cellular replication systems that have become available just recently. 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. We identify a distinct domain in NS5A as the primary “assembly determinant” and show that NS5A and the core protein, which is a major constituent of the virus particle, accumulate on the surface of lipid droplets. Deletions in NS5A disrupting the colocalization of core and NS5A on lipid droplets abrogate infectious HCV production. These studies unravel a unique pathway of infectious virus formation and identify NS5A as a factor modulating HCV replication and assembly and thus viral fitness.