Masters Thesis

Cloning, expression, purification of HPgV (GBV-C virus) NS5B protein – RNA dependent RNA polymerase from inclusion bodies

The GB Virus type – C (GBV-C), formerly known as Hepatitis G Virus/HGV is a member of the Flaviviridae family that has recently been classified under the genus – Pegivirus. It has a positive sense single stranded RNA genome. Among the members of Flaviviridae, most are pathogenic like that of Hepatitis C virus (HCV), West Nile virus, Yellow fever virus and Dengue virus; GBV-C is found to be a non-pathogenic virus. GBV-C is found to infect peripheral blood mononuclear cells (PBMCs), CD4+ and Bcells. The viral RNA dependent RNA polymerase (RdRp) encoded by the NS5B gene, is responsible for replication of the GBV-C genome. Recently, there have been studies showing that co-infection of HIV-1 with GBV-C has shown to slow the replication of HIV-1 and delays the progression to AIDS (Acquired Immune Deficiency Syndrome). However, the mechanism by which GBV-C aids in slowing the replication of HIV-1 is uncertain. Understanding the mechanism of viral replication aids in providing more insight on the viral life cycle and its role in slowing down the HIV-1 replication during co-infection. As mentioned above, most of the members of Flaviviridae are highly pathogenic and understanding the structure and mechanism of the GBV-C polymerase will be useful to study drug target mechanisms and inhibition of replication of other pathogenic members of the family as polymerases have found to be structurally similar. The GBV-C genome possesses nucleic acid sequence similarity with the Hepatitis- C virus (HCV), thus making it a potential model to be used for therapeutic studies since it is a non-pathogenic model. In this Project, the coding sequence of NS5B gene that codes for GBV-C RNA dependent RNA polymerase (RdRp) will be cloned into an expression vector fused with metal binding tag that will allow the purification of the polymerase. The results of this research will potentially aid in obtaining the crystal structure of the protein that can be used for crystallization studies using X-ray diffraction method. Further, the crystallized structure will be used to determine the 3-D structure of the protein. The structure of protein will be helpful in providing a potential non-pathogenic model for therapeutic studies on pathogenic members of the Flaviviridae family by giving more insight on developing inhibitors against drug targets, vaccine development and other clinical research as well as in obtaining a coherent picture of the replication of GBV-C and its role in co-infection with HIV-1.

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