Nonstructural 5A Protein and Transcriptional Activation of Host Genes

26 27 Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is involved in regulating 28 viral replication through its direct interaction with the HCV RNA-dependent RNA 29 polymerase. NS5A also alters infected cell metabolism through complex interactions 30 with numerous host cell proteins. NS5A has furthermore been suggested to act as a 31 transcriptional activator, although the impact on viral replication is unclear. To study 32 this, HCV NS5A variants were amplified from hepatic tissue from an HCV-infected 33 patient, and their abilities to activate gene transcription were analyzed in a single 34 hybrid yeast model. Different variants isolated from the same patient displayed 35 different transactivational activities. When these variants were inserted into the HCV 36 subgenomic replicon system, they demonstrated varying levels of RNA replication, 37 which correlated with their transactivational activities. We showed that the C-terminal 38 fragment of NS5A was localized to the nucleus, and that a functional NS5A nuclear 39 localization signal and cellular caspase activity were required for this process. 40 Furthermore, nuclear localization of NS5A was necessary for viral replication. Finally, 41 we demonstrate that nuclear NS5A binds to host-cell promoters of several genes 42 previously identified as important for efficient HCV RNA replication, inducing their 43 transcription. Taken together, these results demonstrate a new mechanism by which 44 HCV modulates its cellular environment, thereby enhancing viral replication. 45