Hepatitis
C virus (HCV) NS5A protein plays essential roles in both RNA replication and virion
assembly at the surface of cytoplasmic lipid droplets (LDs), although the
precise mechanisms are unclear. In previous work we have shown that tagging
NS5A with a tetracysteine (TCM) motif (Jc1/5A-TCM) and visualising NS5A traffic in a
productive infection results in two distinct NS5A populations: one static and
one highly motile, although the role and composition of these structures is not
well understood. To address if NS5A motile structures contain HCV RNA we
developed a system to simultaneously track HCV RNA and NS5A in living cells. MS2
bacteriophage RNA stem loop sequences (24x) were inserted into the 3’UTR of
Jc1/5A-TCM (Jc1/NS5A-TCM:MS2) virus to allow indirect tracking of HCV RNA in
Huh-7.5 cells via mCherry-tagged MS2 Coat protein that interacts specifically
with MS2 stem loops. Jc1/NS5a-TCM:MS2 replicated to significantly lower levels
than its parent as assessed by infectivity assays (FFA) and immunofluorescence
analysis. However, long-term culture resulted in emergence of viral
replication, with partial retention of MS2 stem loops at 8 days post
electroporation. Most importantly, in these cultures there was a redistribution
of the mCherry tagged MS2 coat protein from a homogenous cytoplasmic
distribution to a punctate staining pattern indicative to specific binding to
HCV RNA. Using this approach we have simultaneously visualised HCV RNA
(MS2coat-mCherry) and NS5A traffic (FlAsH) in real-time during HCV replication.
Interestingly, both NS5A small motile and larger static structures were found
to be enriched with HCV RNA. We also investigated viral RNA traffic with
respect to the lipid droplet (LD) and show that both NS5A static and motile
NS5A enriched structures dynamically interact with the LD consistent with their
role as sites for RNA replication and delivery of HCV RNA to the site of virion
assembly.