Cytoskeleton-associated Pr65 gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells

Abstract

Certain temperature-sensitive ( ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65 gag , at 39°, the nonpermissive temperature. At 39°, virions released from cells infected with the various is mutants also contained elevated levels of Pr65 gag relative to virions released at 33°, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65 gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr659 gag accumulated in the cytoskeleton-enriched fraction at 39° but not at 33°. During steady-state labeling, as much as half of the intracellular Pr65 gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39°. At permissive temperature only 10–15% of the intracellular Pr65 gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65 gag in the cytoskeleton-enriched cell fraction during a short pulse at 39°, but Pr65 gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33°. Based upon these and previous results (Edbauer and Naso,1983), models for retrovirus assembly are described in which the association of Pr65 gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.