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

HCV基因组复制和病毒体形态发生在专用的细胞膜隔室中，所述细胞膜隔室充当执行这些过程的专门蛋白质复合物的组装位点。这些大分子蛋白质复合物的成熟需要蛋白质-蛋白质相互作用的逐步连续，其必须以时空方式严格控制以避免形成非功能性蛋白质复合物。NS 3通过为功能不同的蛋白质复合物的组装提供多用途表面区域在这种时间调节中具有关键作用。通过询问这些NS 3表面区域，我们发现一组遗传上保守的NS 3残基形成了一个连续的表面补丁，跨越蛋白酶和解旋酶结构域，这对NS 5A过度磷酸化至关重要，这一特征对复制酶的形成至关重要，以支持HCV RNA复制。此外，解旋酶残基在这些残基附近是关键的病毒粒子形态发生，而不影响基因组复制。我们提出，NS 3提供了不同的表面区域，以时间和空间的方式调节肝炎病毒生命周期不同步骤中各种专用蛋白质复合物的组装，这些蛋白质复合物对复制酶组装和病毒体形成至关重要。在这项提议中，目的是进一步表征突变引起的缺陷，即与病毒和/或细胞结合配偶体的相互作用被这些突变消除。这些信息将告诉我们RNA复制和病毒体形态发生所需的多蛋白复合物的组成以及它们形成的时空调控。为了揭示干扰复制酶组装或病毒体形态发生的单个突变的影响，我们将遵循两种策略（I）鉴定第二位点突变和（II）鉴定由这些突变诱导的蛋白质复合物组成的变化。

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.