Myristoylated src-oncogene products are potently detected by the monoclonal antibody to the NH2-terminal myristoyl-Gly-Ser-Ser-Lys src-peptide

Monoclonal antibodies were raised against a synthetic NH2-terminal myristoyl tetrapeptide (N-myristoyl-Gly-Ser-Ser-Lys) which is characteristic of an NH2-terminal portion of pp60src, the transforming protein of src-oncogene. The antibody reacted with the albumin conjugated with both the N-myristoyl and N-lauroyl-tetrapeptides, but concentrations at which 50% of the immunoreaction was inhibited were 5 pmol for the N-myristoyl and 830 pmol for N-lauroyl tetrapeptidyl albumin. On the other hand, N-palmitoyl tetrapeptidyl and underivatized albumin, and Gly-Ser-Ser-Lys-Ser-Lys-Pro-Lys octapeptide had no effects. These results suggest a high affinity of the antibody for an N-myristoyl-Gly-Ser-Ser-Lys moiety. src-Oncogene products in Rous sarcoma virus-transformed cells and human colon carcinoma tumor cells were selectively identified as myristoylated pp60src by immunoprecipitation analyses with the antibody.     

Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus

We have used a recombinant vaccinia virus (VV) which expresses high levels of human immunodeficiency virus-1 (HIV-1) gag proteins to analyze the processing pathway of the gag p55 precursor. HIV-1 gag proteins were isolated from [3H]leucine-labeled VV:gag-infected H9 T lymphocytes by immunoprecipitation with either anti-p24, anti-p17, or anti-p6 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that processing of the p55 precursor involves three major intermediates (p41a, p41b, and p39). The p41a and p39 proteins contain the p17 and p24 protein segments, and the p41b is comprised of p24 and p15 segments. On two-dimensional gels, each intermediate as well as the mature p24 and p17 proteins migrated as distinct species. [3H]Myristic acid labeling of the HIV-1 gag proteins revealed that in addition to p55 and p17, the p41a and p39 intermediates, but not p41b, are myristylated, confirming that myristylation occurs at the NH2 terminus before cleavage of the p55 precursor protein. We conclude that the myristylated HIV-1 gag p55 precursor is initially cleaved at random either at the p17/p24 junction or at two sites between p24 and p15 proteins, resulting in three intermediates (p41a, p41b, and p39) which are subsequently cleaved to yield mature gag proteins.     

Neoplastic transformation by E26 leukemia virus is mediated by a single protein containing domains of gag and myb genes.

The genome of avian leukemia virus E26 shares homology with v-myb, the oncogene of avian myeloblastosis virus, and encodes a protein with an Mr of 135,000. Analyses of tryptic oligopeptides show that this protein is related to the proteins encoded by gag (Pr76gag) as well as v-myb (p45v-myb[AMV] ) and c-myb (p75c-myb). We found no evidence for the existence of additional myb-related proteins or subgenomic species of myb-related RNA in myeloblasts transformed by strain E26.     

Analysis of common epitopes on gag proteins of HIV-1, HIV-2 and SIV[AGM] using monoclonal antibodies against HIV-1.

Five monoclonal antibodies (MoAbs) to gag proteins of HIV-1 were prepared in mice. Western blot analyses showed that three clones recognized p24 and the other two p17. Among the three MoAbs recognizing p24, all recognized two of three strains of HIV-2. The spectra of reactions to SIV[AGM] of these MoAbs against p24 were different from one to another; K3-24 recognized all four strains of SIV[AGM], L6-24 three of them, and K5-24 none of them. Of the two MoAbs recognizing p17, K7-17 recognized two of the three strains of HIV-2 but not any SIV[AGM] strain, and the other clone, L14-17 recognized none of analogous proteins of HIV-2 nor of SIV[AGM]. These results demonstrate that the gag proteins of HIV-2 and SIV[AGM] share some common epitopes with those of HIV-1 which are heterogenic in some degree among the different isolates.     

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.     

Immunological characterization of the gag gene products of bovine immunodeficiency virus.

The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus. Gag determinants are also found in the Gag-Pol polyprotein precursor. To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens. Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products. The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively. In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol. Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses. The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins. Based on the present data, a scheme for the processing of BIV Gag precursors is proposed.     

Antigenic specificity of antibody-dependent cell-mediated cytotoxicity directed against human immunodeficiency virus in antibody-positive sera.

Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for human immunodeficiency virus (HIV) has been described for HIV-infected individuals. To determine the antigenic specificity of this immune response and to define its relationship to the disease state, an ADCC assay was developed using Epstein-Barr virus-transformed lymphoblastoid cell line targets infected with vaccinia virus vectors expressing HIV proteins. The vaccinia virus vectors induced appropriate HIV proteins (envelope glycoproteins gp160, gp120, and gp41 or gag proteins p55, p40, p24, and p17) in infected lymphoblastoid cell lines as demonstrated by radioimmunoprecipitation and syncytia formation with c8166 cells. Killer cell-mediated, HIV-specific ADCC was found in sera from HIV-seropositive but not HIV-seronegative hemophiliacs. This HIV-specific response was directed against envelope glycoprotein but was completely absent against target cells expressing the HIV gag proteins. The ADCC directed against gp160 was present at serum dilutions up to 1/316,000. There was no correlation between serum ADCC titer and the stage of HIV-related illness as determined by T-helper-cell numbers. These experiments clearly implicated gp160 as the target antigen of HIV-specific ADCC activity following natural infection. Vaccines which stimulate antibodies directed against gp160, which are capable of mediating ADCC against infected cells, could be important for protection against infection by cell-associated virus.     

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.     

The NH2-terminal amino acid sequence of human proparathyroid hormone by radioisotope microanalysis.

The amino terminal amino acid sequence of human proparathyroid hormone was determined by a highly sensitive radioisotope method. Fresh human parathyroid glands were incubated with one of several ³H-labeled amino acids after which human proparathyroid hormone labeled with [³H]lysine, [³H]serine, [³H]valine, [³H]arginine, or [³H]leucine was isolated. These specimens were subjected to several cycles of Edman degradation. An increase in the amount of radioactivity liberated at any cycle was taken as evidence that the amino acid at that cycle was the one with which the sample was labeled. By this approach, we found that the amino-terminal sequence of human proparathyroid hormone is lys¹-ser²-val³-lys⁴-lys⁵-arg⁶-ser⁷-val⁸-ser⁹ … leu¹³ … leu¹⁷ … lys¹⁹ … Based on these results, we conclude that the amino-terminal region of human proparathyroid hormone consists of a hexapeptide lys-ser-val-lys-lys-arg followed by the amino acid sequence of human parathyroid hormone.     

Transformation of NIH 3T3 cells by DNA of the MCF-7 human mammary carcinoma cell line induces expression of an endogenous murine leukemia provirus.

Previous studies identified two glycoproteins of 86 (gp86) and 72 (gp72) kilodaltons and two nonglycosylated proteins of 70 (p70) and 19 (p19) kilodaltons which were specifically expressed in NIH cells transformed by DNA of the MCF-7 human mammary carcinoma cell line. Pulse-chase experiments and the use of tunicamycin to inhibit glycosylation suggested that gp86, gp72, and p19 were related as precursor products. Characteristics of the four transformation-associated proteins resembled those of murine leukemia virus (MuLV) proteins. Sera raised against disrupted MuLV immunoprecipitated the same four proteins in extracts of NIH(MCF-7) cells and MuLV-infected NIH 3T3 cells. In addition, a monoclonal antibody against MuLV gp70 immunoprecipitated proteins gp86 and gp72, whereas a monoclonal antibody against MuLV p15(E) immunoprecipitated gp86 and p19. These results indicate that proteins gp86, gp72, and p19 expressed in NIH(MCF-7) transformants correspond to MuLV envelope proteins gp80env, gp70, and p15(E), respectively. The transformation-associated protein p70 appears to be a non-envelope MuLV protein, most likely p65gag. Northern blot analysis confirmed that transformation of NIH cells by MCF-7 mammary carcinoma DNA led to the induction of an endogenous MuLV provirus.     

Quantitative separation of murine leukemia virus proteins by reversed-phase high-pressure liquid chromatography reveals newly described gag and env cleavage products.

The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included p10, p12, p15, p30, p15(E), gp69, and gp71 and three previously undescribed virus components designated here as p10', p2(E), and p2(E). Homologous proteins and polypeptides were isolated from M-MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293:543-548, 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of p10' and p2(E)s. The results showed that, during proteolytic processing of gp80env from M-MuLV (M-gp 80env), a single Arg residue was excised between gp70 and p15(E) and a single peptide bond was cleaved between p15(E) and p2(E). The structure of M-gPr80env is gp70-(Arg)-p15(E)-p2(E). The data suggest that proteolytic cleavage sites in R-gp85env are identical to corresponding cleavage sites in M-gp80env. The p2(E)s are shown to be different genetic variants of p2(E) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-Thr-Leu-Asp-Asp-OH. The complete structure of Pr65gag is p15-p12-p30-p10'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, p12, p30, and p10 plus p10') were found in equimolar amounts and p15(E)s were equimolar with p2(E)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Pr15(E) [i.e., p15(E)-p2(E)] is located on the inner side of the lipid bilayer of the virus.     

Comparison of recombinant human immunodeficiency virus gag precursor and gag/env fusion proteins and a synthetic env peptide as diagnostic reagents

Diagnostic reagents for detection of human immunodeficiency virus (HIV) exposure with improved reliability may be provided by viral encoded proteins produced by recombinant DNA techniques or by synthetic peptides corresponding to appropriate viral epitopes. We have expressed at high levels in E. coli a gag gene segment corresponding to approximately 97% of the p55 gag precursor protein, as well as a novel gag/env fusion protein that contains antigenic determinants in common with gag p24, env gp41, and env gp120. The gag and gag/env proteins were purified from insoluble inclusion bodies by sequential extraction with increasing concentrations of urea. These components were tested for reactivity with antisera to HIV proteins and peptides. We have also chemically synthesized a peptide corresponding to env residues 578-608, representing a portion of env gp41. The final preparation of gag and gag/env proteins in 8 M urea reacted with sheep anti-HTLV-III p24 gag antibodies and acquired immune deficiency syndrome (AIDS) patient sera. The gag/env fusion protein also reacted with rabbit anti-HIV env 500-511 peptide antibody. Both recombinant proteins and the env peptide were suitable as reagents for evaluation of serum samples by enzyme-linked immunosorbent assay (ELISA). Results of ELISA assays utilizing the recombinant viral proteins and synthetic peptide were in good agreement with results obtained using disrupted virus as antigen in ELISA assays and immunoblotting.     

The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.     

Feline sarcoma virus-coded polyprotein: enzymatic cleavage by a type C virus-coded structural protein.

A purified 15,000-molecular-weight (Mr) Prague strain Rous sarcoma virus gag gene-coded structural protein, p15, was shown to enzymatically cleave the previously described 130,000 Mr feline sarcoma virus-coded polyprotein, Pr130. Cleavage products included proteins ranging in molecular weight from 12,000 to 110,000. The specificity of this cleavage reactivity was indicated by the fact that, under similar conditions, neither purified type C viral structural proteins nor nonviral proteins such as bovine serum albumin were cleaved to significant extents. Moreover, feline leukemia virus Pr65gag was efficiently cleaved, resulting in the generations of proteins of 30,000 (p30), 15,000 (p15), 12,000 (p12), and 10,000 (p10) Mr. Using enzymatically (p15) treated feline sarcoma virus Pr130 as starting material, we were able to purify a major 72,000 Mr cleavage product and to show it to contain the previously described feline sarcoma virus-coded nonstructural component.     

Chemical and immunological characterizations of equine infectious anemia virus gag-encoded proteins.

The viral core proteins (p15, p26, p11, and p9) of equine infectious anemia virus (EIAV) (Wyoming strain) were purified by reverse-phase high-pressure liquid chromatography. Each purified protein was analyzed for amino acid content, N-terminal amino acid sequence, C-terminal amino acid sequence, and phosphoamino acid content. The results of N- and C-terminal amino acid sequence analysis of each gag protein, taken together with the nucleotide sequence of the EIAV gag gene (R. M. Stephens, J. W. Casey, and N. R. Rice, Science 231:589-594, 1986), show that the order of the proteins in the precursor is p15-p26-*-p11-p9, where a pentapeptide also found in the virus is represented by the asterisk. The data are in complete agreement with the predicted structure of the gag polyprotein and show the peptide bonds cleaved during proteolytic processing. The N-terminus of p15 is blocked to Edman degradation. The p11 protein is identical to the nucleic acid-binding protein of EIAV previously isolated (C. W. Long, L. E. Henderson, and S. Oroszlan, Virology 104:491-496, 1980). High-titer rabbit antiserum was prepared against each purified protein. These antisera were used to detect the putative gag precursor (Pr55gag) and intermediate cleavage products designated Pr49 (p15-p26-*-p11), Pr40 (p15-p26), and Pr35 (p26-*-p11) in the virus and in virus-infected cells. High-titer antisera to EIAV p15 and p26 showed cross-reactivity with the homologous protein of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus.     

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.