Affinity capture and identification of host cell factors associated with hepatitis C virus (+) strand subgenomic RNA

Hepatitis C virus (HCV) infection leading to chronic hepatitis is a major factor in the causation of liver cirrhosis, hepatocellular carcinoma, and liver failure. This process may involve the interplay of various host cell factors, as well as the interaction of these factors with viral RNA and proteins. We report a novel strategy using a sequence-specific biotinylated peptide nucleic acid (PNA)-neamine conjugate targeted to HCV RNA for the in situ capture of subgenomic HCV (+) RNA, along with cellular and viral factors associated with it in MH14 host cells. Using this affinity capture system in conjunction with LC/MS/MS, we have identified 83 cellular factors and three viral proteins (NS5B, NS5A, and NS3-4a protease-helicase) associated with the viral genome. The capture was highly specific. These proteins were not scored with cured MH14 cells devoid of HCV replicons because of the absence of the target sequence in cells for the PNA-neamine probe and also because, unlike oligomeric DNA, cellular proteins have no affinity for PNA. The identified cellular factors belong to different functional groups, including signaling, oncogenic, chaperonin, transcriptional regulators, and RNA helicases as well as DEAD box proteins, ribosomal proteins, translational regulators/factors, and metabolic enzymes, that represent a diverse set of cellular factors associated with the HCV RNA genome. Small interfering RNA-mediated silencing of a diverse class of selected proteins in an HCV replicon cell line either enhanced or inhibited HCV replication/translation, suggesting that these cellular factors have regulatory roles in HCV replication.