The morphogenesis of murine retroviruses involves an initial interaction between two 35s viral RNA genomes and about 2000 Pr65gag polyprotein precursor molecules to form a nucleoprotein complex and the subsequent association of the Pr65gag/RNA complex at the cell surface with lipids and additional envelope proteins to form a nascent, immature particle. Specific proteolytic cleavages of Pr65gag and envelope proteins during and/or after budding by specific proteases then leads to maturation of the virion. We intend to analyze three aspects of this assembly problem: i) The mechanism by which the about 2000 Pr65gag precursor molecules are brought to and then inserted into the plasma membrane. The recent discovery that the NH2 terminus of Pr65gag is myristylated has led us to look at when this modification occurs, e.g., on polysomes? on cytoskeletons? on membranes? Also, what happens to virus production when cerulenin, an inhibitor of fatty acid synthesis, is used? Further, since p15, the NH2 terminal protein on Pr65gag has a stretch of 20 hydrophobic amino acids at its NH2 terminus, we want to know what, if any, is more important for insertion of Pr65gag into the membrane, viz., the myristic acid modification or the presence of about 20 hydrophobic amino acids? Appropriate mutations and deletions in cloned p15 MuLV DNA (corresponding to the NH2 terminus) will be constructed and the DNA will then be used to answer these questions via transfection or microinjection experiments with appropriate host cells. ii) The manner in which the Pr65gag molecules are associated with the cytoskeleton, e.g., is it necessary to be both myristylated and under-phosphorylated? Also, how does this association lead to an alteration of the actin component of the cytoskeleton? iii) The molecular basis by which the """"""""immature"""""""", Pr65gag enriched C-type particles are converted by proteolytic cleavage to """"""""mature"""""""", C-type virus particles. This involves a further study of the murine eetrovirus specific protease activity. Toward this end there now appears good evidence that the protease is virus-coded. It is located on the genome after gag and before pol. We are currently attempting to clone the putative protease gene and isolate the gene product in sufficient quantities, so that the above reaction and concomitant maturation can be characterized in molecular detail.
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