D. R. Taylor1 , M. Puig2 , K. Mihalik3 , E. M. Silberstein1 , S. M. Feinstone3 , 1DETTD, CBER, FDA, Rockville, MD, 2DTP, CDER, FDA, Rockville, MD, 3DVP, CBER, FDA, Rockville, MD
Background: While many clinical hepatitis C virus infections are resistant to interferon alpha (IFN) therapy, subgenomic in vitro self-replicating HCV RNA (HCV replicons), are characterized by marked IFN sensitivity. IFN treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through PKR and also through a new pathway involving RNA editing by adenosine deaminase that acts on double-stranded RNA (ADAR1).
Methods: To measure the effect of IFN on translation, we used a dual-Luc reporter. Replicon-containing cells were cotransfected with either PKR or well-characterized PKR inhibitors, and the Luc reporter. Luciferase activity was measured in cell lysates after IFN-αtreatment. HCV RNA was monitored by real-time RT-PCR. Evidence for ADAR1 dsRNA editing in replicon-containing cells was examined by sequencing of RT-PCR products from cytoplasmic extracts and conversion of radiolabeled AMP to IMP.
Results: We show that inhibition of PKR in replicon-containing cells does not limit the negative effects of IFN. However, inhibition of both PKR and ADAR1 stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold indicating that ADAR1 has a role in limiting replication of the viral RNA.
Conclusions:This is the first report of ADAR's involvement in a potent antiviral pathway and its action to Replicon-containing cells specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful viral clearance by IFN in vitro and may provide a promising new therapeutic strategy to counteract HCV as well as other viral infections.