Characterization of glycosylated Gag expressed by a neurovirulent murine leukemia virus: identification of differences in processing in vitro and in vivo

Abstract

The neuroinvasiveness of a chimeric murine retrovirus, CasFr KP (KP), is dependent on the expression of glycosylated Gag (gp85 gag). This viral protein is the product of alternate translation initiation 88 codons upstream of and in frame with the initiation codon of pr65 gag , the precursor of the viral core proteins. Although expression of glycosylated Gag affects virus spread in the spleen, it appears not to affect virus spread in vitro in fibroblast cell lines (J. L. Portis et al., J. Virol. 68:3879-3887, 1994). The differential effects of this protein in vitro and in vivo have not been explained, and its function is unknown. We have here compared the in vitro processing of this molecule with that expressed in spleens of infected mice. In vitro, gp85 gag was cleaved near the middle of the molecule, releasing the C-terminal half (containing capsid and nucleocapsid domains of pr65 gag) as a secreted glycoprotein. The N-terminal half of the protein was associated with the plasma membrane as a ϳ55-kDa glycoprotein bearing the matrix domain of pr65 gag as well as the N-terminal 88 residue L domain. This processing scheme was also observed in vivo, although two differences were seen. There were differences in N-linked glycosylation of the secreted form of the protein expressed in the spleen. In addition, whereas the membrane-associated species assumed the orientation of a type II integral membrane protein (N cyto C exo) in fibroblasts in vitro, a subpopulation of spleen cells was detected in which the N terminus of the protein was exposed at the cell surface. These results suggest that the differential effects of glycosylated Gag expression in vivo and in vitro may be related to differences in posttranslational processing of the protein.

The Neuroinvasiveness of a Murine Retrovirus Is Influenced by a Dileucine-Containing Sequence in the Cytoplasmic Tail of Glycosylated Gag

“…After the 10-min pulse (time zero), two proteins were labeled: gp85 gag and gPr180 gag-pol , which is a glycosylated gag-pol fusion protein (13). After 10 min of chase, two additional protein appeared: one slightly larger than gp85 gag , which is likely a proteolytic cleavage product of gPr180 gag-pol , and a diffuse protein ranging from 40 to 55 kDa ‫,)ء(‬ which is the N-terminal cleavage product of gp85 gag (15). (B) Quantification of the gp85 gag bands for KP and KP41.…”

“…Lysates were diluted fourfold with a buffer containing 1.25% Nonidet P-40, 1.25% sodium deoxycholate, 0.0125 M sodium phosphate (pH 7.2), 2 mM EDTA, and the protease inhibitors at the concentrations listed above. Lysates were immunoprecipitated as described previously (5) with rabbit antipeptide 4210 (15,29) reactive with the extreme N terminus of glycosylated Gag. Immunoprecipitates were recovered with immobilized protein A (Pierce), eluted by boiling for 5 min in sample buffer containing 2% SDS and 5% 2-mercaptoethanol, and resolved by electrophoresis on 9% polyacrylamide gels (PAGE).…”

“…The latter were grown in tissue culture flasks, from which they were removed with saline-trypsin-EDTA. Infected and uninfected live cells were stained as described previously (15), using the following primary antibodies: monoclonal antibodies 667 (anti-SU) (23) and 34 (anti-Gag MA [matrix] protein) (8) for M. dunni cells and biotinylated monoclonal antibodies 667 and 34 for mouse spleen cells. The mouse primary and biotinylated antibodies were detected by using fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin (Cappel) and fluorescein isothiocyanate-streptavidin (GIBCO), respectively.…”

“…Thus, the coding sequence of glycosylated Gag is identical to that of pr65 gag except for a unique N-terminal sequence, which in the case of the KP virus is 88 residues in length. Unlike pr65 gag , which is a cytosolic protein processed by the viral protease, glycosylated Gag is translated into the endoplasmic reticulum as a type II inte-gral membrane protein (N cyto C exo ), gp85 gag , the C-terminal half being cleaved by a cellular protease and secreted (15). The N-terminal half of the molecule is integrated into the cell membrane by a signal/anchor domain and is displayed at the plasma membrane (15).…”

“…Unlike pr65 gag , which is a cytosolic protein processed by the viral protease, glycosylated Gag is translated into the endoplasmic reticulum as a type II inte-gral membrane protein (N cyto C exo ), gp85 gag , the C-terminal half being cleaved by a cellular protease and secreted (15). The N-terminal half of the molecule is integrated into the cell membrane by a signal/anchor domain and is displayed at the plasma membrane (15). The five amino acids which differ between KP and KP41 are located within the predicted N-terminal cytoplasmic tail.…”