Mason-Pfizer monkey virus (M-PMV), is the prototype exogenous D-type retrovirus. A striking aspect of M-PMV replication is the intracytoplasmic assembly of the capsid shell. These preassembled procapsids then migrate to the plasma membrane where they associate with the glycoprotein- containing lipid bilayer. The major goal of this proposal is to obtain a better understanding of the molecular mechanisms and structural aspects involved in the process of assembling an infectious (D-type) retrovirus particle. The three major aims of this proposal are: To define using genetic and biochemical approaches structural elements in Gag that mediate intracellular transport capsid assembly and plasma membrane association. We will examine the structural basis for intracellular targeting mediated by the cytoplasmic targeting/retention signal, determine the role of matrix protein charged amino acids in membrane association and intracellular targeting, and examine functional aspects of the internal scaffold domain. Furthermore, the ability to assemble capsids in an in vitro translation system allows a direct analysis of regions involved in assembly itself rather than those that might be important for intracellular transport. To identify and define the role of host cell components in the viral assembly/intracellular transport process. We plan to utilize a combination of approaches, including yeast two hybrid systems, gene chip technology, and antibody co-precipitation to identify and characterize novel host-cell components and cellular chaperone proteins involved in the M-PMV capsid assembly/transport process. To determine the role of Env (cytoplasmic domain)/capsid interactions in the process of virus assembly. We plan to define, both structurally and functionally, the role of the cytoplasmic domain of TM in glycoprotein incorporation into M-PMV virions and examine how it regulates the fusion potential of Env. The approaches described above will extend our understanding of the molecular events involved in retrovirus assembly and will pave the way for rational approaches to develop therapeutics that can interfere with these key events in the virus life cycle.
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