Role of cell cytoskeleton in Mo-MuLV env transport and processing: Implications in ts1 neuropathology

Abstract

Treatment of Mo-MuLV-infected cells with cytochalasin B (CB), a microfilament disrupting drug, caused a reduction in virus yield as judged by infectivity assay and reverse transcriptase activity. Pulse-chase experiments with [ 3H]leucine showed that the env precursor, gPr80 env , was inefficiently processed in cells treated with CB. In the presence of monensin, an inhibitor of glycoprotein transport, gPr80 env accumulated intracellularly and no gp70 was observed on the cell surface, indicating a complete block in the processing of gPr80 env . Pulse-chase studies also showed that gPr80 env was not processed in the presence of monensin. SDS-PAGE analysis of TX-100-extracted cell cytoskeletons (TX-insoluble fraction) iodinated and immunoprecipitated with goat anti-gp70 antiserum showed that CB or monensin treatment caused a marked increase of gPr80 env in the cytoskeleton-rich fraction. However, the amount of gPr80 env associated with the TX-soluble fraction in both CB or monensin-treated and untreated cells labeled with [ 3H]leucine was about the same. The gPr80 env in the TX-100-soluble fraction of the cell was the endoglycosidase H (Endo-H) sensitive mannose-rich form, whereas the cytoskeleton-associated gPr80 env was the partially Endo-H-resistant complex carbohydrate form. In the presence of CB or monensin, the complex carbohydrate form of gPr80 env accumulated in the cytoskeleton-rich cell fraction. Examination of Mo-MuLV ts1 mutant, which is defective in the processing of env precursor polyprotein, also revealed an accumulation of the complex carbohydrate form of gPr80 env in the cytoskeleton-rich fraction and an absence of gp70 on the surface of the cell at the restrictive temperature (39°C). These studies suggest that the cytoskeleton plays a role in the transport and processing of MuLV gPr80 env and that oligosaccharide conversion is an important factor in this process. Further, the accumulation of gPr80 env on the cytoskeleton of ts1 infected cells at restrictive temperature may play a role in the neurological disorder caused by Mo-MuLV ts1 mutant.