Characterisation of the membrane and protein interactions of the Hepatitis C Virus NS2

Hepatitis C virus (HCV) readily establishes a chronic infection and it is currently thought to infect 2-3% of the global population. Treatment for HCV places a severe burden on patients, leading to a premature treatment termination in approximately 20% of individuals, and has poor efficacy against the predominant genotype. A greater understanding of the virus lifecycle and mechanisms of persistence will provide valuable information in the continuing development of direct-acting antiviral compounds. HCV encodes 10 proteins which are translated as a single polyprotein. Non-structural protein 2 (NS2) is a small, hydrophobic, trans-membrane protein, yet the precise number and position of its trans-membrane domains remain unclear. NS2 is required for virion morphogenesis but it is not required for replication of the viral genome and it does not form part of the mature virion. NS2 has been shown to interact with other viral proteins, potentially via intra-membrane contacts. Determining the topology of NS2 is therefore vital to our understanding of how NS2 interacts with the other viral proteins and functions within the virus lifecycle. The interactions between NS2 and membranes and the viral glycoprotein E2 were investigated by truncation analysis and fusion with reporter proteins. Glycosylation analysis demonstrated that the N- and C-termini of NS2 are oriented to the luminal and cytosolic faces of the ER membrane, respectively. Truncation of NS2 at residue 70 oriented a C-terminal reporter fusion to the ER lumen consistent with the formation of a luminal loop. This is the first biochemical evidence that NS2 assumes a 3 TMD topology. Co-immunoprecipitation studies with E2 and eGFP-tagged truncation of NS2 revealed that NS2 forms multiple interactions with E2 and that these interactions are dependent upon NS2 targeting to membranes but likely independent of NS2 topology. A model of NS2 topology is presented.