Molecular Determinants of Hepatitis C Virus Replication and Assembly

HCV NS5A expression

Shown here is one of the very first images Dr. Lindenbach took showing that HCVcc is infectious in cell culture (Lindenbach, et al., Science, 2005). Here, HCV NS5A expression (brown staining) can be seen in infected Huh-7.5 cells (blue nuclei).

Based on an HCV infectious clone that replicates to high levels in cell culture, we have been examining the viral and host determinants of RNA replication and virus particle assembly.

Hepatitis C virus genome structure: dynamic roles in replication and infectivity.

In collaboration with Dr. Anna Pyle (HHMI, Yale Department of Molecular, Cellular and Developmental Biology), we have been examining the HCV RNA genome secondary and tertiary structures both in vitro and in vivo, as well as the role of viral proteins in recruiting the viral genome into RNA replication complexes. We have also developed a trans-complementation assay to dissect role(s) for HCV nonstructural (NS) proteins and showed that the NS3 RNA helicase has an essential cis-acting role in assembly of the HCV RNA replication complex. We are currently following up on the hypothesis that the helicase acts to divert viral RNA into different functional compartments, including RNA replication.

Molecular determinants of hepatitis C virus assembly.

We discovered that the HCV NS2 protein, in addition to encoding a self-cleaving protease, has an essential role in virus particle assembly, bringing together the viral E1-E2 glycoprotein complex and the RNA replication complex. We have further examined the hypothesis that the NS3 helicase contributes to virus assembly by acting as a motor protein to package the viral genome.

Funding: R01 AI087925, R01 AI131518