The long term goal of our studies is to examine the interactions that retroviral structural (Gag) proteins make with each other and with other viral components during virus morphogenesis, when particles undergo a major reorganization from immature to mature forms. The Moloney murine leukemia virus (M-MuLV) Gag proteins form the major internal core struCtures of M-MuLV particles, and accumulated evidence indicates that appropriate Gag protein interactions with the virus lipid envelope. the viral genomic RNA, other Gag proteins, and products of the pol and env genes are crucial to M-MuLV replication. Our focus concerns the mechanism by which Gag proteins oligomerize to form virions, and we will analyze the structures of Gag monomers, and the interactions made between them. These studies are essential to the characterization of retrovirus core proteins and particles; address basic unresolved questions conCerning all enveloped animal viruses; and will be needed for optimization of retrovirus vectors for gene therapy purposes, and to guide the design of rational core-targeted antiretroviral compounds. We have developed novel biophysical and molecular approaches for virus structure determination, and will employ these methods in our investigations. The procedures we describe should lead to an understanding of retrovirus core interactions, and should be applicable for analysis of a wide variety of proteins.
Our specific aims are as follows: 1. Electron-diffraction image analysis of retroviral Gag proteins: Histidine-tagged (his-tagged) retrovirus Gag proteins will be used to make two dimensional protein arrays on phosphatidyl choline (PC) monolayers containing novel nickel-chelating lipids. Stained protein and unstained arrays will be viewed by transmission electron microscopy (EM) to obtain data concerning Gag protein interactions, and to obtain 5-10 angstrom resolution maps by image enhancement-electron diffraction methods. This approach will complement standard procedures and may be developed for analysis of virtually any his-tagged protein. 2. Analysis of Gag protein interactions in retrovirus particles: Molecular genetic methods will be used to examine Gag protein interactions in virions. Deletion mutants of gag matrix and nucleocapsid domains will be characterized to evaluate the contributions of these regions to final particle structures. Using a unique cysteine-minus (cys- ) parental M-MuLV construct. cysteine-substitution mutants will be analyzed in crosslinking and chemical reactivity studies. Additionally, a cellular protein which crosslinks to the retroviral Gag protein major homology region (MHR) will be characterized. These studies will complement biophysical analyses, and will be used to test predictions based on biophysical data and molecular models.
