Characterisation of functional domains in the NS2-3 cysteine protease and the NS3-4A of hepatitis C virus and their role in the assembly of the viral replicase

丙型肝炎病毒基因组复制和病毒粒子形态形成发生在专门的细胞膜隔室中，这些隔室充当执行这些过程的专门蛋白质复合体的组装地点。这些大分子蛋白质复合体的成熟需要一个循序渐进的蛋白质-蛋白质相互作用序列，这些相互作用必须以时空的方式严格控制，以避免形成无功能的蛋白质复合体。NS3通过为功能不同的蛋白质复合体的组装提供多用途表面积，在这种时间调控中发挥着关键作用。通过询问这些NS3表面区，我们发现一组在系统发育上保守的NS3残基在蛋白酶和解旋酶结构域之间形成了一个连续的表面补丁，这对NS5A过度磷酸化是关键的，这一特征被证明是复制酶形成的重要特征，以支持丙型肝炎病毒RNA复制。此外，靠近这些残基的解旋酶残基对于病毒粒子的形态发生至关重要，而不会影响基因组复制。我们认为，在肝炎病毒生命周期的不同阶段，NS3提供了不同的表面积来以时间和空间的方式调节各种特定蛋白质复合体的组装，这些蛋白质复合体对复制酶组装和病毒粒子的形成至关重要。在这个建议中，目的是进一步表征由突变引起的缺陷，即这些突变取消了哪些与病毒和/或细胞结合伙伴的相互作用。这些信息将使我们了解核糖核酸复制和病毒粒子形态形成所需的专用多蛋白质复合体的组成(S)及其形成的时空规律。为了揭示单个突变干扰复制酶组装或病毒粒子形态形成的影响，我们将遵循两种策略(I)鉴定第二位点突变和(Ii)鉴定由这些突变引起的蛋白质复合体组成的变化。

HCV genome replication and virion morphogenesis take place in dedicated cellular membrane compartments that serve as assembly sites of specialized protein complexes executing these processes. Maturation of these macromolecular protein complexes requires a step-wise succession of protein-protein interactions that must be tightly controlled in a spatio-temporal manner to avoid the formation of non-functional protein complexes. NS3 has a pivotal role in this temporal regulation by providing multipurpose surface areas for the assembly of functionally distinct protein complexes. By interrogating these NS3 surface areas, we show that a set of phylogenetically conserved NS3 residues forms a continuous surface patch across protease and helicase domain that is critical for NS5A hyperphosphorylation, a feature shown to be important for replicase formation to support HCV RNA replication. Furthermore, a helicase residue in close proximity to these residues is pivotal for virion morphogenesis without affecting genome replication. We propose that NS3 provides distinct surface areas to regulate, in a temporal and spatial fashion, the assembly of various, dedicated protein complexes during the different steps of the hepaciviral life cycle that are crucial for replicase assembly and virion formation.In this proposal, the aim is to further characterize the defects caused by the mutations, i.e. which interactions with viral and/or cellular binding partners are abrogated by these mutations. This information will inform us on the composition(s) of dedicated multi-protein complexes required for RNA replication and virion morphogenesis and the temporal and spatial regulation of their formation.To unravel the effect of the individual mutations either interfering with replicase assembly or virion morphogenesis we will follow two strategies (I) identification of second site mutations and (II) identifications of changes in protein complex composition induced by these mutations.