A rapid screening procedure for the isolation of nonconditional replication mutants of Mason-Pfizer monkey virus: identification of a mutant defective in pol.

A rapid, sensitive, and reproducible method for the isolation of human cell clones containing nonconditional, replication-defective (rd) mutants of Mason-Pfizer monkey virus (M-PMV), the prototype of the D-type retroviruses is described. The two mutants, rd1 and rd2, thus far isolated have been analyzed for virus particle production (using radiolabeled precursors and by electron microscopy) and for the status of intracellular viral precursors. Thin sections of rd1 and rd2 infected cells showed typical M-PMV particles when observed under electron microscope. A more direct assay of virus production, by labeling the mutant cell clones with [3H]uridine, also showed a distinct virus peak at an approximate density of 1.16 g/ml when culture fluids from rd1 and rd2 were analyzed. Analyses of these two mutants showed no defect in either gag or env gene products, however, further analysis of rd1 showed that the Pr180gag-pol was altered in its migration on SDS-polyacrylamide gel electrophoresis and no reverse transcriptase activity could be detected in rd1 virions. Mutant rd2, on the other hand, assembles noninfectious virus particles that are otherwise indistinguishable from those produced by wild-type cell clones. The biochemical basis for the defect in this mutant remains to be established.