The Molecular Mechanism Of The Role Of GPS2in Hepatitis C Virus Replication

As a growing public health concern, Hepatitis C virus (HCV) infects about170million people worldwide and frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Current treatment options for chronic hepatitis C are limited and accompanied with some side effects. Thus, there is an urgent need to develop effective therapeutic strategies against this human pathogen.HCV is a single-stranded and positive sense RNA virus within the family Flaviviridae. Its genome encodes a single precursor polyprotein composed of about3,000amino acid (aa), which is cleaved by both cellular and viral proteases to generate three structural (core, E1, and E2) and seven nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. NS5A is an essential component of the HCV replicase and also strictly required for HCV assembly, while no known enzymatic function has been ascribed to it. NS5A exists as multiple phospho-isoforms and contains three discrete domains following the N-terminal membrane-anchoring a-helix. It interacts with a wide variety of host cell proteins, such as FK506-binding protein8(FKBP8) and vesicle-associated membrane protein-associated protein A (VAP-A, also called hVAP-33), and exerts a wide range of effects on cellular pathways and processes, including innate immunity and host cell growth and proliferation.Recently, G protein pathway suppressor2(GPS2) has been found to interact with HCV NS5A in a high-throughput yeast two-hybrid (Y2H) screens of human cDNA library using viral proteins as baits. GPS2is a ubiquitous protein that was originally identified while screening for suppressors of Ras activation in the yeast pheromone response pathway. Several studies have indicated the role of GPS2in transcriptional regulation through interacting with a series of transcriptional regulators, such as the NCoR/SMRT nuclear receptor corepressor complex. Meanwhile, a critical cytoplasmic role for GPS2in inhibiting the proinflammatory TNF-a pathway is also uncovered. Here we first confirmed this interaction both in vitro and in physiological condition using co-immunoprecipitation analysis. Mutagenesis analysis revealed that domains I of NS5A and N-terminal coiled-coil domain of GPS2are responsible for their interaction. Using retroviral-based RNA interference assay, we demonstrated that GPS2is reqiured for HCV replication. Moreover, we found that GPS2is needed for efficient association between NS5A and VAP-A, which is critical for the formation of HCV replication complex and HCV RNA replication. We demonstrated that overexpression of GPS2facilitates the association of NS5A with VAP-A, while knockdown of GPS2obviously impairs NS5A association with VAP-A. These results together indicate that GPS2acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.