Exploring genetic and physical aspects of Hepatitis C Virus non-structural proteins involved in the formation of the replication complex

Hepatitis C Virus (HCV) is the causative agent of Hepatitis C, a chronic liver disease that is responsible for significant morbidity and mortality throughout the world. Replication of the HCV RNA genome relies on the expression of non-structural proteins (NS), produced initially as a single polyprotein. Often subgenomic viral transcripts that express the NS polyprotein are used to study RNA replication. Previous work in the laboratory led to the development of a novel ‘intragenomic’ replicon, a construct expressing two copies of the NS polyprotein, thus readily enabling genetic complementation studies to be undertaken. Using this system, it was shown that genetic defects in NS4B and NS5A could be experimentally separated on the basis of the minimum polyprotein required to rescue these defects; some mutations were dependent on NS3-NS4A-NS4B-NS5A (NS3-5A) but others required NS3-NS4A-NS4B-NS5A-NS5B (NS3-5B). In this project, we investigated if this categorization could be extended to mutations elsewhere in the NS polyprotein, focussing on the viral protease/helicase NS3, as well as its co-factor NS4A. Three mutations were identified whose rescue could be facilitated by NS3- 5A; one in the NS3 linker region (PP1220-1GG), another in the NS3 protease domain (L1157A), and a third in the NS4A acidic domain (Y1706A). In contrast, one mutation in the NS4A transmembrane domain (V1665G) could not be rescued by NS3-5A. Furthermore, this same mutation exhibited a dominant negative phenotype, but consistent with the model of polyprotein–dependent complex formation, only when expressed in the context of NS3- 5B, but not when expressed in the context of NS3-4A or NS3-5A. An investigation into the mechanisms governing this difference in polyprotein rescue found that many of the mutations typically dependent on NS3-5B for rescue could be rescued by NS3-5A if this latter polyprotein was linked to a downstream coding region. This suggested that nascent NS3-5A was making interactions critical to replication complex formation while tethered to the genome via partially translated NS5B. In a separate study looking at the role that NS proteins play in remodelling lipid bilayers, the interaction of NS4B with neutral and anionic lipids was assessed by static and magic angle spinning (MAS) 31P nuclear magnetic resonance (NMR) spectroscopy. Static NMR showed that NS4B caused a broadening of the powder pattern of POPC multilamellar vesicles (MLVs) and that, at high temperature (45°C), there is significant bilayer disturbance with loss of the axially symmetric pattern. The MAS NMR showed that in the presence of anionic lipids, NS4B leads to changes in the surface charge density. This suggests that NS4B interacts with anionic lipids and that this interaction changes the bilayer mobility. These changes were also confirmed by measuring the T1 relaxation time.