This project uses analytical ultracentrifugation (AU), light-scattering, and other biophysical analytical tools for the characterization of viral protein interactions and other protein interacting systems. This year we have focused on four research areas: (1) VLP Assembly. HIV particle assembly is an essential step in the viral replication cycle and is a potential target for antiviral therapy. However, molecular mechanisms in assembly are not understood as yet. The major structural component in all retrovirus particles is the Gag protein. Gag is a multi-domain protein that is cleaved during virus maturation into a series of cleavage products. The immature virus particle contains full length Gag. Alan Rein's laboratory has discovered that a virus like particle (VLP) can be generated by adding nucleic acid to Gag and assembly cofactors. We are currently performing a rigorous biophysical analysis of the molecular events underlying VLP assembly. This analysis will focus on (a) detection of Gag oligomers as potential assembly intermediates; (b) interactions of Gag protein with the assembly cofactors; and (c) conformational changes in Gag protein molecules. (2) L1R Viral Protein. Bernie Moss' Laboratory of Viral Diseases has been studying all aspects of the vaccina virus replication cycle over a very extended period of time. The structure of poxvirus virions is complex and the outer surface, unlike that of most other viruses, lacks either helical or icosahedral symmetry. Intriguingly, however, immature virions (IVs) are completely enclosed by a honeycomb lattice, the formation and fate of which are poorly understood. Last year we presented the results of biochemical, microscopic and genetic studies demonstrating that the D13 protein of vaccina virus exists as trimers that assemble to form the external honeycomb lattice of IVs. The trimerization state was determined by both gel electrophoretic and analytical ultracentrifuge (AU) methodologies. Live vaccinia virus (VV) was used as a vaccine to eradicate smallpox (variola virus) worldwide. However, there is still concern that remaining variola virus stocks could be used as a biological weapon. Mass vaccination with VV is one solution to this threat but its severe side effects pose a risk to certain segments of the population. Another solution is to generate a subunit vaccine. The viral protein L1R is synthesized late in infection and is incorporated into intracellular mature virions (IMV). It also plays an essential role in virus assembly. Blocking of L1R expression prevents incorporation of DNA into virions. Importantly, L1R is a major target of VV neutralizing antibodies. Based on the above considerations it was of considerable importance to provide a full characterization of L1R. For this characterization we have again used AU to establish that L1R behaves as a monomeric protein. (3) Recombinant Proteins. The Malaria Vacccine Development Branch of NIAID is exploring the use of recombinant Plasmodium falciparum surface proteins for their potential to vaccinate against malaria. We are assisting the characterization of selected proteins using AU methodologies. (4) Light-scattering Methodologies. A development project is in progress with the Physical Biochemistry Section of NIDDK led by Allen Minton. The latter has developed light-scattering methodology for the determination of association constants. To promote the utilization of this approach to other binding interactions, we are duplicating the instrumentation in our DBEPS Molecular Interactions Resource and studying a number of systems, such as the VLP assembly of the HIV gag protein described above.
