Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles

The hepatitis C virus (HCV) NS5A protein plays a critical role in viral RNA replication and has recently been shown to play a role in particle production in the infectious genotype 2a HCV clone (JFH-1). Here, we show that alanine substitutions of serines 2428/2430 within the C-terminal domain III of NS5A do not affect subgenomic replicon RNA replication but do reduce particle production. In contrast, substitution of serines 2390/2391 had no effect on either RNA replication or particle production. Relative to genotype 1, all genotype 2 HCV isolates contain a 19 residue insertion near the C terminus of domain III which, when deleted (Delta 2408-2426), resulted in a delay to both RNA replication and particle production. None of these mutations affected the ratio of basal to hyperphosphorylated NS5A, suggesting that serines between residues 2390 and 2430 are not phosphorylated. We propose that although domain III is dispensable for RNA replication, it nevertheless influences this process.