To determine if the cellular factors La autoantigen (La) and polypyrimidine tract-binding protein (PTB) are required for hepatitis C virus (HCV) replication, we used siRNAs to silence these factors and then monitored their effect on HCV replication using quantitative RT-PCR. In addition, we determined the influence of PTB on the activity of the 3′ noncoding region (NCR) of HCV and investigated its interaction with the components of the HCV replicase complex. We found that La is essential for efficient HCV replication while PTB appears to partially repress replication. PTB does, however, block the binding of HCV RNA-dependent RNA polymerase (RdRp, NS5B) to the 3′NCR. Indirect immunofluorescence microscopy showed co-localization of cytoplasmic PTB with the HCV RdRp in hepatoma cells (Huh-7) expressing HCV proteins, while in vitro translation of viral proteins from the HCV replicon revealed the interaction of PTB isoforms with NS5B polymerase and NS3.
Bibliographical noteFunding Information:
We are highly thankful to M.M.C. Lai for pThNS5B, C.M. Rice for BB7 replicons, C. Seeger for the FCA4 cell line, S. Das for pET21a-HCV NS3, and G. Dreyfuss for monoclonal anti-PTB antibody (7G12). N.A. was supported by an American Liver Foundation Ira M. Jacobson MD Liver Scholar Award. This work was supported by an NIH grant (DK061566) to A.S. and an NIH grant (T32 A107537-03) to A.D.
- HCV replication
- HCV translation