Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV.     

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.     

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.     

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.     

Role of a cytotoxic-T-lymphocyte epitope-defined, alternative gag open reading frame in the pathogenesis of a murine retrovirus-induced immunodeficiency syndrome

LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60gag). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.     

Hierarchal Utilization of Different T-Cell Receptor Vβ Gene Segments in the CD8+-T-Cell Response to an Immunodominant Moloney Leukemia Virus-Encoded Epitope In Vivo

The CD8(+)-T-cell response to Moloney murine leukemia virus (M-MuLV)-associated antigens in C57BL/6 mice is directed against an immunodominant gag-encoded epitope (CCLCLTVFL) presented in the context of H-2D(b) and is restricted primarily to cytotoxic T lymphocytes (CTL) expressing the Valpha3.2 and Vbeta5.2 gene segments. We decided to examine the M-MuLV response in congenic C57BL/6 Vbeta(a) mice which are unable to express the dominant Valpha3.2(+) Vbeta5.2(+) T-cell receptor (TCR) due to a large deletion at the TCR locus that includes the Vbeta5.2 gene segment. Interestingly, M-MuLV-immune C57BL/6 Vbeta(a) mice were still able to reject M-MuLV-infected tumor cells and direct ex vivo analysis of peripheral blood lymphocytes from these immune mice revealed a dramatic increase in CD8(+) cells utilizing the same Valpha3.2 gene segment in association with two different Vbeta segments (Vbeta3 and Vbeta17). Surprisingly, all these CTL recognized the same immunodominant M-MuLV gag epitope. Analysis of the TCR repertoire of individual M-MuLV-immune (C57BL/6 x C57BL/6 Vbeta(a))F(1) mice revealed a clear hierarchy in Vbeta utilization, with a preferential usage of the Vbeta17 gene segment, whereas Vbeta3 and especially Vbeta5.2 were used to much lesser extents. Sequencing of TCRalpha- and -beta-chain junctional regions of CTL clones specific for the M-MuLV gag epitope revealed a diverse repertoire of TCRbeta chains in Vbeta(a) mice and a highly restricted TCRbeta-chain repertoire in Vbeta(b) mice, whereas TCRalpha-chain sequences were highly conserved in both cases. Collectively, our data indicate that the H-2D(b)-restricted M-MuLV gag epitope can be recognized in a hierarchal fashion by different Vbeta domains and that the degree of beta-chain diversity varies according to Vbeta utilization.     

Further studies on the glycosylated gag gene products of Rauscher murine leukemia virus: identification of an N-terminal 45,000-dalton cleavage product

A glycosylated 45,000-Mr protein containing Rauscher murine leukemia virus p15 and p12 antigenic sites and tryptic peptides was identified in Rauscher murine leukemia virus-infected cells. This glycoprotein, termed gP45gag, was also shown to contain a single tryptic peptide also present in gPr80gag and its unglycosylated apoprotein precursor Pr75gag, but lacking in Pr65gag or Pr40gag. The presence of this peptide only in viral precursor proteins containing the so-called leader (L) sequence strongly suggests that gPr45gag is an N-terminal fragment of larger glycosylated gag polyproteins, composed of L sequences in addition to p15 and p12. The kinetics of appearance of radiolabeled gPr45gag and its disappearance during chase-incubation is suggestive of a precursor-like role for this intermediate gene product. An observed 27,000-Mr glycosylated polypeptide, termed gP27gag and containing p15 but not p12, p30, or p10 antigenic determinants, is a candidate cleavage product derived from gPr45gag. These observations suggest that gPr45gag and its putative cleavage product gP27gag represent an authentic pathway for intracellular processing of glycosylated core proteins.     

N myristylation of the human immunodeficiency virus type 1 gag polyprotein precursor in Saccharomyces cerevisiae.

A semisynthetic gene precisely encoding the 502 amino acids of the human immunodeficiency virus type 1 gag precursor (Pr53gag) was expressed in the yeast Saccharomyces cerevisiae. Amino acid sequence analysis of the recombinant Pr53gag showed that the amino terminus was fully blocked. Labeling of Pr53gag with [3H]myristic acid demonstrated that, as with Pr53gag isolated from virus-infected cells, the yeast-derived protein was demethionylated and N myristylated on glycine, the second amino acid residue.     

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.     

Identification of a new viral protein containing CAp30 and NCp10 sequences in murine and feline leukemia retroviruses.

Because Pr65gag is in part located in the nucleus and contains a putative bipartite nuclear targeting signal, we investigated the cellular location and structure of P55gag, a gag-encoded polyprotein known to lack the nucleocapsid (NC) protein NCp10. P55gag was found to be restricted to the cytoplasm of Moloney murine leukemia virus-infected cells. Of interest, P55gag was produced in cells infected by a viral protease deletion mutant and by a recombinant murine sarcoma virus known to lack the protease gene. Surprisingly, our structural and immunological studies indicated that P55gag also lacks carboxy-terminal residues of CAp30. During the course of studying P55gag, we detected a new viral protein within purified virus particles that contained NCp10 tryptic peptide sequences and a CAp30 tryptic peptide lacking in P55gag. This viral protein, which we have named nucleocapsid-related protein (NCRP), also contained antigenic epitopes present in CAp30 and NCp10. P55gag- and NCRP-like proteins were also observed in AKV murine leukemia virus and feline leukemia virus systems. The precise site of cleavage within Pr65gag that produces P55gag and NCRP is unknown but lies upstream of the CAp30-NCp10 junction within the carboxy-terminal domain of CAp30. The existence of a form of NCp10 containing carboxy-terminal CAp30 sequences raises interesting possibilities about its functional role in genomic RNA packaging and/or viral RNA dimerization.     

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.     

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.     

Cytotoxic T lymphocytes do not appear to select for mutations in an immunodominant epitope of simian immunodeficiency virus gag.

Studies to date assessing HIV escape from CTL in vivo have yielded conflicting results. Previous studies have demonstrated that simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys expressing the MHC class I allele Mamu-A*01 reproducibly develop a gag-specific CTL response limited to a 9-amino acid epitope of the SIVmac gag protein (residues 182-190 within peptide 11C). To determine whether CTL have a role in selecting for AIDS virus mutants, we examined mutations in SIVmac proviral DNA encoding this gag CTL epitope in PBL of infected rhesus monkeys. Three Mamu-A*01+ rhesus monkeys were infected with SIVmac and assessed for gag- and peptide 11C-specific CTL responses. This specific CTL response was maintained in two monkeys, but lost in the third animal 2 yr after infection. The generation of proviral gag mutations was then determined by sequencing 500-bp proviral fragments amplified from fresh PBL obtained from the monkeys more than 2.5 yr after infection. Although numerous point mutations were characterized in 131 polymerase chain reaction-generated clones of SIVmac gag, only four mutations within the gag CTL epitope-coding region of the genome were identified. Comparison of synonymous and nonsynonymous nucleotide substitutions in the regions encoding peptide 11C (p11C) and the flanking gag protein indicated a lack of selective pressure for viral mutations in the CTL epitope coding region. Interestingly, a predominant gag mutant encoding a single amino acid change in p11C was found in a monkey which lost its CTL activity. However, even in this setting there was no evidence for selection of mutations in the CTL epitope coding region when compared with the flanking region. Furthermore, synthetic peptides corresponding to all naturally occurring variants in the gag epitope-coding region were recognized by cloned and bulk cultured effector cells of the infected monkeys with persistent CTL. These results indicate that SIVmac gag- and p11C-specific CTL do not select for mutations in the immunodominant epitope-coding region and that the naturally occurring mutants do not appear to escape CTL recognition.     

An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag.

A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone. This epitope is highly conserved among 18 HIV-1 strains. The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2. This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli. Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24. The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag. This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.     

Identification of a novel tumor-specific CTL epitope presented by RMA, EL-4, and MBL-2 lymphomas reveals their common origin

C57BL/6 mice generate a vigorous H-2Db-restricted CTL response against murine leukemia virus (MuLV)-induced tumors. For many years it has been suggested that this response is directed to an MuLV-encoded peptide as well as to a nonviral tumor-associated peptide. Recently, a peptide from the leader sequence of gag was demonstrated to be the MuLV-derived epitope. Here we describe the molecular identification of the tumor-associated epitope. Furthermore, we show that the CTL response against this epitope can restrict the outgrowth of MuLV-induced tumors in vivo. The epitope is selectively presented by the MuLV-induced T cell tumors RBL-5, RMA, and MBL-2 as well as by the chemically induced T cell lymphoma EL-4. Intriguingly, these tumors share expression of the newly identified epitope because they represent variants of the same clonal tumor cell line, as evident from sequencing of the TCR alpha- and beta-chains, which proved to be identical. Our research shows that all sources of RBL-5, RMA, RMA-S, MBL-2, and EL-4 tumors are derived from a single tumor line, most likely EL-4.     

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.     

gag-Related polypeptides encoded by replication-defective avian oncoviruses.

The content of viral structural (gag) protein sequences in polypeptides encoded by replication-defective avian erythroblastosis virus (AEV) and myelocytomatosis virus MC29 was assessed by immunological and peptide analyses. Direct comparison with gag proteins of the associated helper viruses revealed that MC29 110K polypeptide contained p19, p12, and p27, whereas the AEV 75K polypeptide had sequences related only to p19 and p12. Both of these polypeptides contained some information that was unrelated to gag, pol, or env gene products. In addition, no homology was detected between these unique peptides of MC29 110K and AEV 75K. The AEV 75K polypeptide shared strain-specific tryptic peptides with the p19 encoded by its naturally occurring helper virus; this observation suggests that gag-related sequences in 75K were originally derived from the helper viral gag gene. Digestion of oxidized MC29 110K and AEV 75K proteins with the Staphylococcus aureus V8 protease generated a fragment which comigrated with N-acetylmethionylsulfoneglutamic acid, a blocked dipeptide which is the putative amino-terminal sequence of structural protein p19 and gag precursor Pr76gag. This last finding is evidence that the gag sequences are located at the N-terminal end of the MC29 110K and AEV 75K polypeptides.     

In vivo modification of retroviral gag gene-encoded polyproteins by myristic acid.

It has recently been shown by mass spectral analysis (Henderson et al., Proc. Natl. Acad. Sci. U.S.A. 80:339-343, 1983) that the p15gag protein of murine leukemia viruses contains a novel post-translational modification, an amino-terminal myristyl (tetradecanoyl) amide. In this report we show that p15gag is the only structural protein to contain this fatty acid. In addition, the gag precursor polyproteins of type B, C, and D retroviruses have been examined for the presence of myristic acid by metabolic labeling and immunoprecipitation studies. In a panel of mammalian type C retroviruses we found that the precursor polyprotein Pr65gag homologs, but not the glycosylated forms (gPr80gag homologs), were specifically labeled after a 5-min incubation of infected cells with [3H]myristic acid. The gag precursor polyprotein was also labeled in mouse mammary tumor virus and in Mason-Pfizer monkey virus, but Pr76gag of Rous sarcoma virus failed to incorporate [3H]myristate. Under similar conditions, [3H]palmitate was not found to be incorporated into any viral gag proteins. Thus, myristylation appears to be a common feature of mammalian type B, C, and D retroviruses but not of avian retroviruses.     

Biochemical and immunological analysis of human immunodeficiency virus gag gene products p17 and p24.

Human immunodeficiency virus (HIV) p24 was purified to homogeneity and subjected to NH2-terminal sequencing. The sequence determined perfectly corresponded to the amino acid sequence predicted from the nucleotide sequence of a middle portion of the HIV first open frame: the gag gene. Edman degradation of purified HIV p17 revealed instead a blocked NH2 terminus. Hybridomas secreting monoclonal antibodies to p24 and p17 were developed and used to immunologically characterize these two HIV gag gene products. They identified two gag precursor polyproteins in the cytoplasm of HIV-infected cells: Pr53gag, which corresponds to the primary translational product, and Pr39gag, which corresponds to an intermediate product of cleavage of Pr53gag. These monoclonal antibodies allowed us also to study posttranslational modification of HIV p24 and p17. p24 was found to be phosphorylated, which is a very unusual feature for a major retroviral core protein. p17 was found to be myristylated, as are all NH2-terminal gag proteins of the known human retroviruses.