Topology and membrane rearrangements of the hepatitis C virus protein NS4B

Sammanfattning: Hepatitis C virus (HCV) is a major cause of chronic liver disease and viral hepatitis around the world. Approximately 180 million people or 3% of the world's population are infected with the virus. The infection does only rarely give rise to any acute symptoms, but more than 70% of those infected develop chronic hepatitis with the risk of developing liver cirrhosis and liver cancer. The only treatment available is a combination therapy of pegylated interferon and the broad spectrum antiviral agent ribavirin. All patients cannot be subjected to this treatment however, and of those treated only about 50% (with variations for different genotypes) will clear the virus. A better understanding of HCV and its life cycle is therefore needed to achieve a more efficient and well tolerated therapy against HCV infection. In order to improve the understanding of HCV, we have focused on one of the least known of the viral proteins - the non structural protein 4B (NS4B). NS4B is a relatively small protein (27 KDa) of mostly unknown functions. It is believed however to play an important, but yet unknown part in the viral replication process. By employing fluorescence microscopy we found that NS4B located in the endoplasmic reticulum (ER) membrane, and that it also had the ability to rearrange the membranes into dense aggregates in the cytoplasm. These new structures are called membrane associated foci (MAF) and are believed to be the locale for viral replication. They are assumed to be the same structures, or closely related, as the structure called "membraneous web" found with electron microscopy. In our studies we also found that NS4B is an integral ER membrane protein. By computer predictions and glycosylation mapping we generated a model where the protein initially has four transmembrane segments with the C- and the N-termini in the cytoplasm. After translation and processing from the polyprotein however, the N-terminus of NS4B translocates to the ER lumen, giving the protein a fifth transmembrane segment. In the presence of another viral protein, NS5A, a lower degree of translocation was observed, indicating that NS5A may influence the dual topology of NS4B. Immunoprecipitation showed us that NS4B could homo-oligomerize, and this ability seemed to correlate with induction of MAF structures. Furthermore, we obtained experimental data suggesting that the translocation of the N-terminus may be involved in this process too and in particular an amphipatic helix around aa 40-69. Finally, based on our topology model we created 14 point mutations in the NS4B protein in the subgenomic replicon to assess what portions of NS4B that are important for replication. All mutants except one had negative effects on the replicon establishment efficiency, even changes located to the luminal loops of NS4B (replication occurs on the cytoplasmic side). Neither the polyprotein efficiency nor the ability to induce MAF were affected, supporting a role of NS4B in HCV replication besides providing the necessary environment for viral replication.