Membrane interface specific reactions in viral pathogenesis

Wider research context / theoretical framework Protein scaffold of enveloped viruses assembles via matrix protein adsorption on the membrane of a host cell. Interaction of matrix proteins with lipid membranes was investigated for the last decades. It was implied that (1) the lateral lipid distribution is uniform. Even when the non-uniform distribution of anionic lipid was suggested to be crucial, the convincing experimental data supporting it were missing; and that (2) the scaffold assembly and disintegration are at thermodynamic equilibrium, assuming both lateral distribution of matrix protein on the membrane and pH profile normal to the membrane uniform. In existing in vitro experimental data on the membrane adsorption of M1 from Influenza A (M1-A), the mere adsorption was thought to be leading to the formation of the virus-like protein scaffold, even though, unlike a viral scaffold, such M1 layers failed to perform bending of the membrane or budding from it, as well as to respond by disintegration to the acidification of the media. It demonstrates that not every adsorbed M1 layer is virus-like, and in principle an inadequacy of the existing scaffold formation paradigm for Influenza A. Hypotheses / research questions / objectives Comparing structure and the known details of scaffold assembly process of matrix proteins from Influenza A and C, as well as from Ebola viruses suggests that lateral lipid heterogeneity plays an important role, especially for M1-A. We propose (1) to investigate the anionic lipid clustering, in particular of phosphatidylinositols, and what it depends on in the context of viral replication, as well as the role of such clustering in the process of M1-A scaffold formation, and (2) to find the conditions for pH-triggered disassembly of the scaffold in vitro, in particular by mimicking the non-equilibrium proton distribution during the acidification of the virion. Approach / methods We will use both recombinant and purified M1-A, a combination of high resolution structural and optical methods and model lipid bilayers (free-standing, supported and giant unilamellar vesicles) of controlled content and asymmetry. Level of originality / innovation For the first time, the scaffold formation process will be assessed in vitro with a system mimicking the cellular cytoplasmic membrane, both in terms of the lipid asymmetry and small (nanoscale) domain size. For the first time, we will test if the obtained protein structures are virus-like by checking if they respond by disintegration to pH drop. This will allow to provide a comprehensive physical model of viral scaffold assembly and disintegration. Primary researchers involved Dr. Denis Knyazev, Dr. Oleg Batishchev