The role of phosphorylation of the hepatitis C virus NS5A protein revealed by a combination of biochemistry and reverse genetics

Hepatitis C virus causes a chronic infection that affects 2-3 % of the world population and is a significant causative agent of liver cirrhosis and hepatocellular carcinoma. It has a positive-sense, single-stranded genome that encodes for a single polyprotein that is cleaved by both host and viral proteases into 10 mature viral proteins. The non-structural 5A (NS5A) protein is a pleiotropic protein with essential functions in the replication and production of virus, as well as in perturbing host pathways in favour of virus persistence. NS5A is extensively phosphorylated, forming two distinct species termed the basally and hyperphosphorylated; however the location and function of phosphorylation remains poorly defined. To address this, a significant quantity of NS5A was purified from an actively replicating context and subjected to extensive phospho-mapping by mass spectrometry. Through this approach 12 phosphorylation sites were identified. Subsequent phenotyping of phosphorylation sites by reverse genetics generated evidence for a sequential phosphorylation cascade within the low complexity region I, and that this was responsible for the formation of the hyperphosphorylated species. Further analysis identified the phosphorylation of the distant serine 146 as negatively regulating formation of the hyperphosphorylated species. To investigate phosphorylation events in greater detail the SNAP-tag technology (NEB) was applied to NS5A. The insertion of the SNAP-tag into domain III of NS5A was shown to be well tolerated with respect to virus replication and function of the SNAP-tag. In parallel this study also set out to establish whether domain II of NS5A had a hitherto unknown function in the production of infectious virus, however no such effects were observed. In combination with existing data it is clear that no residue within NS5A domain II is essential for the production of infectious virus. Furthermore, this work highlights significant differences in the requirement for domain II in virus replication between different HCV genotypes, despite high conservation within this region.