Mutation in the glycosylated gag protein of murine leukemia virus results in reduced in vivo infectivity and a novel defect in viral budding or release

Abstract

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80gag) in addition to the polyprotein precursor of the viral capsid proteins (Pr65gag). gPr80gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65gag. The role of glyco-Gag in MuLV replication has been unclear, since gPr80gag-negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.

FIG. 1.

gPr80^gag and Ab-X-M-MuLV. The relationship of the coding sequences for Pr65^gag and gPr80^gag is shown. gPr80^gag is translated from the same reading frame as Pr65^gag, but from an upstream CUG initiation codon. The location of the stop codon present in the genome of Ab-X-M-MuLV is shown (X); it is upstream from the Pr65^gag AUG codon.

FIG. 2.

Establishment of in vivo infection by Ab-X-M-MuLV. Neonatal NIH Swiss mice were inoculated intraperitoneally with wild-type M-MuLV or Ab-X-M-MuLV. At different days postinoculation, the animals were sacrificed, and single-cell suspensions were prepared from bone marrow, thymus, and spleen. The levels of infection (number of infectious centers [ICs]/10⁶ cells plated) are shown. Each point represents the result for one animal. Shaded boxes, wild-type M-MuLV (43D cells); solid diamonds, Ab-X-M-MuLV (17-5 cells). (A) Bone marrow; (B) thymus; (C) spleen.

FIG. 3.

AFM of infected cells. AFM was performed on NIH 3T3 cells infected with wild-type M-MuLV (43D) or Ab-X-M-MuLV (17-5). (A) Uninfected NIH 3T3 cell; (B to D) wild-type M-MuLV-infected 43D cell at three magnifications; (E to G) Ab-X-M-MuLV-infected 17-5 cell at three magnifications. The widths of the fields shown are indicated in micrometers. The white bars indicate the widths of the particles. The surface viral structures are typical of infected cells and absent from uninfected NIH 3T3 cells, as described previously (13).

FIG. 4.

AFM of PA317/BAG cells. AFM of the Am-MuLV-based retroviral vector packaging cell line PA317 is shown. These cells were stably transfected with the BAG vector plasmid and produced infectious BAG vector. (A to C) Surface of a PA317/BAG cell at three magnifications. (D to F) Surface of a PA317/BAG cell expressing gPr80^gag (PA317/BAG 8065-2) at three magnifications. Note the presence of spherical particles instead of tubes.

FIG. 5.

gPr80^gag expression plasmid. (A) Organization of the gPr80^gag expression plasmid p8065-2. The CUG initiation codon has been converted to an AUG codon; the backbone expression plasmid is pZeoSV. (B) Western blotting of PA317/BAG versus PA317/BAG 8065-2, using two antibodies raised against the N terminus of gPr80^gag (a gift of John Portis) (9). The location of gPr80^gag is indicated.

FIG. 6.

Vector release from PA317/BAG cells. (A) PA317/BAG cells were transfected with the gPr80^gag expression plasmid p8065-2 or the backbone plasmid pZeoSV ΔMCS, and the cells were grown in medium containing Zeocin to select for stable transfectants. After 2 weeks (when selection was complete), supernatants were collected from the cultures and assayed for production of infectious BAG vector as described in Materials and Methods. The cultures were passaged for an additional 4 weeks in the presence of Zeocin, and the amounts of infectious BAG vector released were again assayed. The titrations were performed with four replicates for each time point. (B) The amount of vector particles released from transfected cultures at the 6-week time point was determined by SDS-PAGE and Western blotting for viral CA (p30) protein. The amount of Gag protein in the cell extracts is also shown; samples representing equal amounts of cells were loaded in all lanes.

FIG. 7.

Virus release by metabolic labeling. PA317, PA317/BAG + ΔMCS, and PA317/BAG + 8065-2 cells, as described in the legend to Fig. 6, were labeled with [³⁵S]methionine-cysteine by a 15-min pulse-chase (A to C), 60-min pulse-chase (D to F), or continuous labeling (G and H). One-half of the released virus or cell lysate for each sample was analyzed by SDS-PAGE. The lengths of the chases or continuous labels are indicated in hours at the top of each panel (0 to 4 h); cell lysates (cell) are shown in the left four lanes, and released viruses (sup) are shown in the right four lanes. The locations of Pr65^gag and cleaved CA protein in released virus are indicated. The more slowly migrating radioactivity in the cell lysates is nonspecific binding; a partial proteolytic cleavage product of Pr65^gag is evident in the cell lysates as well (6). gPr80^gag is evident in the cell lysate from the 15-min pulse (*); during the chase, this protein was glycosylated to form higher-molecular-weight products (6). Autoradiographic exposures were done for 2 weeks at −80°C.