Preparations enriched for “immature” murine leukemia virus particles that remain in tissue culture fluids are deficient in Pr65 gag proteolytic activity

Abstract

Partially purified Moloney or Rauscher murine leukemia virus preparations obtained from 24-hr harvests of tissue culture fluids were separated into Pr65 gag enriched and Pr65 gag deficient fractions by centrifugation on shallow sucrose or sucrose-D 2O density gradients, e.g., 30 to 50% sucrose (w/v). The Pr65 gag enriched fraction had a slightly higher relative density (ϱ = 1.18–1.20 g/cc) and a greatly increased number of “immature” virions (30%), than the Pr65 gag deficient fraction (ϱ = 1.16–1.18 g/cc; 2% immature virions). These two morphologically distinct virus populations were then further characterized as to the state of the RNA, the relative amount of exogenous reverse transcriptase activity, and the presence or absence of Pr65 gag proteolytic activity. Both types of particles possessed 70 S RNA and similar amounts of exogenous reverse transcriptase activity. However, only the Pr65 gag deficient particles exhibited any Pr65 gag proteolytic activity when incubated with Nonidet P-40 (2%, w/v), 10 m M DTT, and a Pr65 gag substrate, i.e., immature virions. Thus it appears that the immature Rauscher and Moloney leukemia virions that remain in tissue culture fluids of chronically infected cells possess a cleavable Pr65 gag, but are deficient in Pr65 gag proteolytic activity.