Assembly and composition of intracellular particles formed by Moloney murine leukemia virus.

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.     

High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay.

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.     

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

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.     

Expression of the gag-pol fusion protein of Moloney murine leukemia virus without gag protein does not induce virion formation or proteolytic processing.

A mutant of Moloney murine leukemia virus was generated in which the UAG termination codon at the 3' end of the gag gene was changed to a CAG codon encoding glutamine. Cells carrying the mutant provirus constitutively express the gag-pol fusion protein and no detectable gag protein. The precursor is stable, is not processed by the protease domain within the precursor, and does not induce assembly and release of virion particles.     

Characterization of the gag/fusion protein encoded by the defective Duplan retrovirus inducing murine acquired immunodeficiency syndrome.

Murine acquired immunodeficiency syndrome is induced by a defective retrovirus. Sequencing of this defective viral genome revealed a long open reading frame which encodes a putative gag/fusion protein, N-MA-p12-CA-NC-COOH, (D. C. Aziz, Z. Hanna, and P. Jolicoeur, Nature (London) 338:505-508, 1989). We raised a specific antibody to the unique p12 domain of this gag fusion precursor, Pr60gag. We found that Pr60gag was indeed encoded by the defective viral genome both in cell-free translation reticulocyte extracts and in infected mouse fibroblasts. Pr60gag was found to be myristylated, phosphorylated, and attached to the cell membrane, like other helper murine leukemia virus (MuLV) gag precursors. Pr60gag was not substantially cleaved within the nonproducer cells and was not released from these cells. However, in the presence of helper MuLV proteins, it formed phenotypically mixed particles. In these particles, Pr60gag was only partially cleaved. In helper MuLV-producing cells harboring the defective virus, a gag-related p40 intermediate was generated both intracellularly and extracellularly. In these cells, Pr60gag appeared to behave as a dominant negative mutant, interfering with proper cleavage of helper Pr65gag. Our data indicate that Pr60gag is a major (and possibly the only) gene product of the defective murine acquired immunodeficiency syndrome virus and is likely to harbor some determinants of pathogenicity of this virus.     

Characterization of intracellular precursor polyproteins of Moloney murine leukemia virus.

Intracellular Moloney murine leukemia viral precursor polyproteins were compared with mature viral proteins by immunoprecipitation and tryptic peptide mapping experiments. The results were consistent with precursor roles for Pr65gag, Pr200gag-pol, Pr135pol, and gPr83env. The glycosylated gag gene product gPr85gag, although containing sequences characteristic of all four core proteins plus additional sequences not found in Pr65gag, lacked a major tyrosine-containing p30 tryptic peptide, suggesting that gPr85gag is not processed to p30.     

Requirements for incorporation of Pr160gag-pol from human immunodeficiency virus type 1 into virus-like particles.

The roles of the human immunodeficiency virus precursor polyproteins Pr55gag and Pr160gag-pol in viral core assembly were studied in CMT3-COS cells. To do this, the precursors were expressed separately by using a simian virus 40 late replacement vector system described previously. Consistent with previously published data, our results show that the Pr55gag precursor, when expressed alone, was able to form particles which had an immature morphology and that particle formation required the presence of a myristate addition signal at the amino terminus of the precursor. In contrast, the Pr160gag-pol precursor was not able to form particles when expressed alone, although it still underwent proteolytic processing. Coexpression of the two precursor polyproteins from separate vectors in the same cell resulted in processing of the Pr55gag in trans by the protease embedded in Pr160gag-pol and the formation of virus-like particles containing the products of both precursors. Proteolytic processing occurred independently of the presence of a functional myristate addition signal on either precursor. On the other hand, removal of myristate from one or the other precursor had nonreciprocal effects on virus particle formation. Cells expressing Pr55gag lacking myristate and Pr160gag-pol containing it did not produce particles. Cells expressing a myristylated Pr55gag and unmyristylated Pr160gag-pol still produced virus-like particles which contained nearly normal amounts of Pr160gag-pol. The results suggest that the incorporation of Pr160gag-pol into particles is largely determined by intermolecular protein-protein interactions between the two precursor polypeptides.     

Unmyristylated Moloney murine leukemia virus Pr65gag is excluded from virus assembly and maturation events.

The gag precursor polyprotein of Moloney murine leukemia virus (MuLV) is normally modified by myristylation of the N-terminal glycine. Previous work showed that the Pr65gag lacking the myristylation site does not associate with cellular membranes or assemble into virus particles. We now report that it also is not cleaved to the mature gag cleavage products within the cell and that it sediments as a free 65-kilodalton monomer in detergent-free cell extracts containing 0.3 M NaCl. Even when the cells containing the mutant are productively infected with wild-type MuLV, the mutant Pr65gag is not processed into cleavage products and is not incorporated into the virions produced by these cells. Thus, the mutant gag molecules seem unable to participate in the normal processes of self-assembly and maturation. We propose that myristate-mediated membrane association is an essential first step in MuLV assembly. This association may also play a role in budding of MuLV.     

Specific inhibitor of human immunodeficiency virus proteinase prevents the cytotoxic effects of a single-chain proteinase dimer and restores particle formation.

The active form of the retroviral proteinase (PR) is a homodimer of monomeric subunits expressed as integral parts of the viral gag-pol precursor polyproteins, and dimerization of polyproteins is presumed to be important for regulation of PR activity. Expression of a single-chain dimer of the human immunodeficiency virus (HIV) type 1 PR as a component of the viral polyprotein has been shown to prevent particle assembly and viral infectivity (H.-G. Kr?usslich, Proc. Natl. Acad. Sci. USA 88:3213-3217, 1991). Ro31-8959, a specific inhibitor of HIV PR, blocked proteolysis of polyproteins containing either wild-type or single-chain dimer PR at the same inhibitor concentration. Different inhibitor concentrations gave three phenotypic effects for the linked PR: at a concentration of 10 nM, cytotoxicity was prevented yet viral polyproteins were almost completely processed and no particles were released. The majority of HIV capsid proteins was found in the soluble cytoplasmic fraction, whereas at a concentration of 1 microM inhibitor most HIV gag proteins were associated with an insoluble fraction. Release of particles consisting of partially processed polyproteins was observed at 100 nM Ro31-8959, and polyprotein processing was blocked at 10 microM. Particles derived from the dimer-containing provirus were noninfectious independently of the inhibitor concentration. Production of infectious HIV after transfection of wild-type provirus was abolished at 100 nM and markedly reduced at 10 nM Ro31-8959.     

Cloned mouse mammary tumor virus DNA exhibits glucocorticoid-dependent expression in simian virus 40-transformed mink cells.

Mink lung epithelial cells were transfected with two cloned mouse mammary tumor virus (MMTV) DNAs, a 9-kilobase clone derived from an unintegrated exogenous viral genome and a 14-kilobase clone containing an integrated endogenous provirus along with cellular flanking sequences. Mink lung cells were chosen because they do not contain endogenous MMTV sequences. On the basis of our observation that simian virus 40 DNA efficiently transforms these cells, we isolated cell clones containing MMTV DNA by using transformation with simian virus 40 DNA as a selective marker in cotransfection experiments. Levels of the 9-kilobase MMTV mRNA representing the entire viral genome and of the spliced 4.4-kilobase mRNA which codes for the viral envelope proteins were glucocorticoid dependent in transformed cells. Expression of low levels of Pr77gag, the precursor of the group-specific viral core proteins, and of gPr73env, the precursor of the viral envelope proteins, was also hormone dependent. We conclude that these cloned MMTV DNAs contain all the information necessary for the synthesis of normal viral RNAs and proteins. These findings also provide further evidence that the DNA sequences involved in the hormone responsiveness of MMTV expression are contained within the viral genome.     

Polyprotein Precursors to Mouse Mammary Tumor Virus Proteins

Mouse mammary tumor virus (MMTV) derived from the culture medium of GR cells contained seven proteins, identified as gp55, gp33, p25, pp20, p16, p12, and p10. The major viral phosphoprotein was the 20,000-molecular-weight protein, pp20. Immunoprecipitation of cytoplasmic extracts from pulse-labeled GR cells identified three MMTV gag-specific proteins, termed Pr78(gag), Pr110(gag), and Pr180(gag+). These intracellular polyproteins were precipitable from cytoplasmic extracts by antisera to virions p25 and p12 but not by antisera to gp55. The major intracellular gag-specific precursor polyprotein, Pr78(gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, p10, and presumably pp20. This precursor is presumably derived from nascent chain cleavage or rapid posttranslational cleavage of the larger intracellular precursor-like protein, designated Pr110(gag). Pr110(gag) contained all but one of the leucine-containing tryptic peptides of Pr78(gag), plus several additional peptides. In addition to Pr78(gag) and Pr110(gag), monospecific antisera to virion p12 and p25 were also capable of precipitating from pulse-labeled cells a small amount of a 180,000-molecular-weight precursor-like protein, designated Pr180(gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78(gag) and Pr110(gag) plus several additional peptides. By analogy to type C viral systems, Pr180(gag+) is presumed to represent a gag-pol common precursor which is the major pathway for synthesis of MMTV polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two env-specific proteins, designated gPr76(env) and gP79(env). The major env precursor, gPr76(env), could be labeled with radioactive glucosamine and was shown to contain antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A minor glycoprotein, gP79(env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79(env) represents fucosylated gPr76(env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.