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.     

Myristylation of Pr60gag of the murine AIDS-defective virus is required to induce disease and notably for the expansion of its target cells.

Murine AIDS (MAIDS) is characterized by severe lymphadenopathy and splenomegaly. The proliferation of the infected target B cells is also an important manifestation of the disease (M. Huang, C. Simard, D. G. Kay, and P. Jolicoeur, J. Virol. 65:6562-6571, 1991). The etiologic agent of MAIDS is a defective murine leukemia virus that is deleted of most of its pol and env genes and appears to encode a single protein, the Gag precursor Pr60gag protein. Pr60gag is myristylated and attached to the plasma membrane. To study the role myristylation on the function of Pr60gag, we have generated a myristylation-negative (Myr-) mutant of the MAIDS defective virus. We found that Myr- Pr60gag interacted less tightly with the plasma membrane. In addition, the Myr- MAIDS defective virus mutant was unable to induce expansion of infected cells and was nonpathogenic. These results emphasize the essential role of Pr60gag in the disease process. Our data also suggest that Pr60gag, once recruited to the cell membrane through its myristylation, interacts with other membrane-bound effectors to send signals to induce proliferation of the infected cells and to initiate immune dysfunctions.     

CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus

Murine leukemia virus (MuLV) codes for two precursors of the group-specific antigens, Pr65gag and Pr75gag, in vivo. While Pr65gag is the precursor to the virion structural proteins, Pr75gag undergoes glycosylation and is found on the surface of the infected cell as gp85gag, and it is thought to play a role in virus maturation and spread. Pr65gag synthesis starts at an AUG codon within a favourable initiation context (AAUAUGG at positions 618 to 624). The gp85gag start codon is upstream but its precise location is not known. To map the initiation codon of gp85gag, we used deletion and site-directed mutagenesis of the leader sequence of MuLV RNA and in vitro translation of the RNAs. Synthesis of the MuLV gp85gag protein appears to be initiated at a CUG codon located within a favourable context (ACCCUGG at positions 354 to 359 for Moloney-MuLV). The possible function of gp85gag was investigated by expressing Moloney-MuLV and Friend-MuLV proviral DNA and mutants deficient for gp85gag synthesis in mouse and rat cells. The results indicate that the gp85gag protein probably facilitates the spread of virus infection in tissue culture.     

The nonstructural component of the Abelson murine leukemia virus polyprotein P120 is encoded by newly acquired genetic sequences.

The Abelson leukemia virus (AbLV) polyprotein P120 is compared to translational products representing the entire Moloney murine leukemia virus (MuLV) genome on the basis of [35S]methionine tryptic peptide composition. Three methionine-containing tryptic peptides present in Moloney Pr65gag are each shown to be present in both Pr75gag and in Pr180gag-pol. Of these, one peptide, corresponding to Moloney MuLV p12, but neither of two p30-specific peptides are present in AbLV P120. Among the 12 remaining methionine-containing peptides present in AbLV P120, many, if not all, are unique to AbLV P120 and not shared by either Moloney MuLV Pr180gag-pol or Pr82gag.     

The GAG precursor of simian immunodeficiency virus assembles into virus-like particles.

To examine the potential role of the GAG precursor polyprotein in morphogenesis and assembly of the simian immunodeficiency virus (SIV), we have expressed the gag gene of SIVMac using a baculovirus expression vector. Infection of insect cells with recombinant virus containing the entire gag gene results in high expression of the GAG precursor protein, Pr57gag. The recombinant protein is myristylated and is released in the culture supernatant in an insoluble particulate form. A point mutation in the N-terminal glycine codon (Gly----Ala) inhibits myristylation. This mutated product is highly expressed but is not found in the culture supernatant. Electron microscopy and immunogold labelling of infected cells show that the native Pr57gag protein assembles into 100-120 nm virus-like particles that bud from the cell plasma membrane and are released in the culture supernatant. The unmyristylated protein also assembles into particulate structures which only accumulate inside the cells. These results demonstrate that the unprocessed GAG precursor of SIV can spontaneously assemble into particles in the absence of other viral proteins. Myristylation of the Pr57gag precursor is necessary for its association with the cell plasma membrane, for budding and for extracellular release.     

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.     

Sequence relationship of glycosylated and unglycosylated gag polyproteins of Moloney murine leukemia virus.

Both glycosylated and unglycosylated polyproteins coded by the gag gene are produced in cells infected with Moloney murine leukemia virus. GpP80gag is a glycosylated precursor of a larger gag glycoprotein exported to the cell surface, whereas Pr65gag is an unglycosylated precursor of the virion internal structural proteins. GpP80gag contains not only carbohydrate, but also additional polypeptide sequences not found in Pr65gag. In the experiment reported here, we localized the differences between GpP80gag and Pr65gag with respect to the domains of the individual gag proteins. This was done by comparison of partial proteolytic cleavage fragments from Pr65gag, from GpP80gag, and from the unglycosylated form of GpP80gag (P75gag) which had been immunoprecipitated by antisera specific for gag proteins p30, p15, and p10. We conclude that the additional polypeptide sequences in GpP80gag are located at or very near the amino terminus of the polyprotein. The carbohydrate in GpP80gag is attached to polypeptide sequences held in common between GpP80gag and Pr65gag.     

Inhibition of murine leukemia virus Pr65gag cleavage in vitro and in vivo by hypertonic medium.

Cleavage of murine leukemia virus Pr65gag is associated with the activity of a labile proteolytic factor found in virions. We have shown that the presence of 80 to 100 mM NaCl inhibits this cleavage activity in vitro by over 90%. Further, the addition of 80 to 100 mM excess NaCl in vivo to chronically infected cultures of MJD-54 mouse fibroblasts also caused inhibition of Pr65gag cleavage. Specifically, the excess salt added to cells: (i) caused a greater than 90% decrease in virus production; (ii) increased the Pr65gag/p30 ratio in virions produced by more than threefold; and (iii) in pulse-chase experiments, showed a 10-fold decrease in the amount of Pr65gag cleaved after 3 h. In contrast, during this chase interval there was only a slight diminution, i.e., about two fold, in the cleavage of env precursor polyprotein Pr80env, suggesting that cleavages of Pr65gag and Pr80env are differently controlled. Additionally, electron microscopic examination of the excess salt-treated cells showed a twofold increase in the number of associated immature particles, consistent with the observed higher than average Pr65gag/p30 ratio. The inhibitory effects were also found if excess KCl or MgCl2 was used instead of NaCl, suggesting that they are caused by the hypertonic state of the medium and are not dependent on the ionic species used.     

A murine leukemia virus mutant with a temperature-sensitive defect in membrane glycoprotein synthesis.

Cells infected with a temperature-sensitive mutant (ts-26) of Rauscher murine leukemia virus (R-MuLV) or with wild-type virus were labeled with 35S-methionine, and cell extracts were examined for radioactive polypeptides which could be precipitated by monospecific antisera to viral proteins. When shifted from permissive (31 degrees C) to nonpermissive (39 degrees C) temperature, cells infected with ts-26 rapidly begin to accumulate gPr90enr, the glycoprotein precursor to the membrane envelope glycoprotein gp70 and to the membrane-associated protein p15E. Simultaneously, formation of these mature virion proteins ceases. In addition, lactoperoxidase-catalyzed surface labeling with 125I--iodine indicates that the plasma membrane of cells infected with ts-26 becomes depleted of gp70 antigens at 39 degrees C. Nevertheless, at 39 degrees C these cells release defective MuLVs which lack gp70 and p15E but contain an outer membrane. The released particles also contain an aberrantly processed form of the major virion core protein p30, and many of these virion cores have an unusual immature crescent shape. It has previously been reported that cells infected with the ts-26 mutant of R-MuLV process a 65,000 dalton precursor (Pr65gag) of the virion core proteins more slowly at 39 degrees C than do cells infected with wild-type virus (Stephenson, Tronick and Aaronson, 1975). Although we have confirmed these results, this effect is relatively small and it is known that various alterations of MuLV assembly can lead secondarily to inhibited processing of Pr65gag. We propose that the ts-26 mutant has a primary temperature-sensitive defect in membrane glycoprotein synthesis and that this change causes pleiotropic effects on core morphogenesis.     

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.     

Post-translational modification of Rauscher leukemia virus precursor polyproteins encoded by the gag gene.

Post-translational modifications of retrovirus gag gene-encoded polyproteins include proteolytic cleavage, phosphorylation, and glycosylation. To study the sequence of these events, we labeled JLS-V9 cells chronically infected with Rauscher murine leukemia virus in pulse-chase experiments with the radioactive precursors [35S]methionine, [14C]mannose, [3H]glucosamine, and [32P]phosphate. Newly synthesized gag polyproteins which incorporated label, and the modified products derived from them, were identified by immunoprecipitation of cell lysates with anti-p30 rabbit serum, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Pulse-chase experiments were carried out in the presence as well as in the absence of tunicamycin, an inhibitor of glycosylation. Among the three major polyproteins synthesized in the absence of tunicamycin, two were found to be glycosylated but not phosphorylated. These were designated gPr80gag and gP94gag. Both shared identical [35S]methionine peptides with Pr65gag and p30. Of the two nonglycosylated precursors, Pr65gag and Pr75gag, only Pr65gag was found to be detectably phosphorylated, and Pr75gag could be readily identified only when glycosylation was inhibited. On the basis of these results, a scheme for the post-translational modification of gag polyproteins is proposed. According to this scheme the gag gene-encoded polyproteins are processed from a common precursor, Pr75gag, by two divergent pathways: one leading through the intermediate Pr65gag to internal virion components via cleavage and phosphorylation and the other via tunicamycin-sensitive mannosylation to the intermediate gPr80gag, which is further glycosylated to yield cell surface polyprotein gP94gag.     

Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus

Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences.     

Localization of lipid-protein and protein-protein interactions within the murine retrovirus gag precursor by a novel peptide-mapping technique

In HTG2 hamster cells infected with the replication-defective Gazdar murine sarcoma virus only immature virus particles are formed, with the uncleaved gag precursor Pr65 as the only major protein in the virion. We have investigated the structure of these particles by using in situ cross-linking followed by chemical and enzymatic cleavages of Pr65 to localize sites of lipid-protein and protein-protein interactions. Lipid-protein cross-links were localized within a 10-kDa fragment in the p15 region of Pr65. Homotypic protein-protein cross-links between Pr65 units were localized within the p15 regions and also within the p10 regions of Pr65. Similar data for processed gag proteins in Rauscher murine leukemia virus, a prototype of a mature C-type virus, suggest that these interactions of the gag precursor are not altered during maturation. To identify the sites of cross-linking within Pr65, we have developed a two-dimensional peptide mapping technique that is based on nearest neighbor analysis of fragments released by cyanogen bromide treatment of partial cleavage products in gel slices. In conjunction with cross-linking, the peptide mapping technique is a powerful means for localizing specific interactions on a polypeptide backbone.     

Spleen focus-forming virus: specific neutralization by antisera to certain gag gene-encoded proteins.

Immunization of rats with syngeneic cells infected with spleen focus-forming virus (SFFV) but not with its helper, Friend murine leukemia virus (FMuLV), produces antisera which specifically neutralize SFFV, and not FMuLV, in the Friend virus complex. To determine which SFFV-encoded protein molecule bears the antigen recognized by these neutralizing antibodies, we studied different lots of rat anti-SFFV antiserum by immunoprecipitation and virus neutralization assays. The ability of these sera to neutralize SFFV correlated with the titer of antibodies to p45gag and not with the titer of those to gp52, suggesting that the neutralizing antibodies recognize the p45gag molecule. To verify this specificity for p45gag, we tested antisera to various MuLV gag gene-encoded proteins for neutralization of SFFV. Goat anti-Rauscher murine leukemia virus (RMuLV) p30 and goat anti-RMuLV p10 sera neither precipitated p45gag from SFFV-infected nonproducer cells nor neutralized SFFV. In contrast, goat anti-RMuLV Pr65gag and goat anti-RMuLV p12 sera precipitated p45gag from SFFV-infected cells and also specifically neutralized SFFV in the Friend virus complex. These findings suggest that, unlike the gag proteins coded for by FMuLV, the proteins coded for by defective SFFV are incorporated into the envelope of virions carrying the SFFV genome, but not into the envelope of those carrying the helper FMuLV genome.     

Creation and expression of myristylated forms of Rous sarcoma virus gag protein in mammalian cells.

Rous sarcoma virus (RSV), a member of the avian sarcoma and leukosis family of retroviruses, has long been known to be capable of infecting and transforming mammalian cells; however, such transformed cells do not release virus particles. The RSV gag product (Pr76gag) produced in these cells is not released into the culture medium or proteolytically processed to release mature products. Thus, the behavior of Pr76gag in mammalian cells is much like that of mammalian retroviral Gag proteins which have been altered so as to block the addition of myristic acid at residue 2 (Gly). Because the RSV gag product does not possess a myristic acid addition site, we hypothesized that the creation of one by oligonucleotide-directed mutagenesis might permit particles to be released from mammalian cells. Two myristylated forms of Pr76 were created. In Pr76myr1, the first 10 amino acids have been exchanged for those of p60v-src, which are known to be sufficient for myristylation. In Pr76myr2, the Glu at the second residue has been substituted with Gly. The alleles encoding the modified and wild-type forms of Pr76 have been expressed at high levels in mammalian (CV-1) cells by using an SV40-based vector. Surprisingly, we have found that expression of high levels of the unmodified (wild-type) product, Pr76myr0, results in low levels of particle formation and precursor processing. This indicates that myristic acid is not the sole determinant for targeting. However, the addition of myristic acid to Pr76myr1 or Pr76myr2 resulted in a fivefold enhancement in Gag function. In all aspects examined, the behavior of myristylated Pr76 was identical to that of the authentic product produced in avian cells. We also show that processing is mediated by the gag-encoded protease and that removal of the amino terminus to create Pr76gagX results in an inability to form particles or be processed. This suggests that proper targeting is prerequisite for activation of the RSV protease in mammalian cells.     

Rous sarcoma virus expression in Saccharomyces cerevisiae: processing and membrane targeting of the gag gene product.

In avian cells, the product of the gag gene of Rous sarcoma virus, Pr76gag, has been shown to be targeted to the plasma membrane, to form virus particles, and then to be processed into mature viral gag proteins. To explore how these phenomena may be dependent upon cellular (host) factors, we expressed the Rous sarcoma virus gag gene in a lower eucaryote, Saccharomyces cerevisiae, and studied the behavior of the gag gene product. We show here that Pr76gag is processed in yeast cells and that this processing is specific, since it is abolished in a mutant in which the active site of the gag protease has been destroyed. In this mutant, the uncleaved precursor is found associated with the yeast plasma membrane, yet no virus particles were detected in cells or in the culture medium. From our results, we can speculate either that in yeast cells, a host protease initiates Pr76gag processing in the cytosol or that in avian cells, an inhibitor prevents the processing until the viral particle is formed.     

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.