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We have introduced GFP and photoactivatable GFP into the NS5A coding region of a hepatitis C virus (HCV) subgenomic replicon that gives efficient transient replication. NS5A-GFP, expressed by the replicon, could be detected in cytoplasmic fluorescent foci as early as 4 h after RNA was introduced into cells. The fluorescent foci are likely to be sites where RNA synthesis could occur, although their production was not dependent on prior replication. Photobleaching studies demonstrated that the fluorescent proteins were relatively immobile upon expression from replicon RNAs. By contrast, an NS5A-GFP chimera produced in the absence of other viral proteins was mobile. Hence, interactions in cells expressing HCV replication proteins limit NS5A mobility, and transfer of viral proteins between foci is either slow or does not occur. Thus, the sites of HCV RNA replication possibly have a fixed complement of proteins that may act as discrete factories for producing viral RNA.

Mobility analysis of an NS5A–GFP fusion protein in cells actively replicating hepatitis C virus subgenomic RNA