Suppression of the respiratory burst of human monocytes by a synthetic peptide homologous to envelope proteins of human and animal retroviruses.
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Humans↗
Biomedical subjects
T D Copeland
Publications and source records attributed to T D Copeland.
C-terminal region of human T cell lymphotropic virus type I (HTLVI) p19 core protein is immunogenic in humans and contains an HTLVI-specific epitope.
To study the human host response to viral structural proteins during HTLV type I infection, five synthetic peptides matching the N-terminal and C-terminal regions of HTLVI p19 core protein were used to identify antigenic sites on p19 that were immunogenic in man. In radioimmunoassay and immunoprecipitation experiments, antibodies in 16 of 18 HTLVI+ patient sera reacted with a synthetic peptide matching the C-terminal 11-amino acid sequence of p19, whereas only two sera contained antibodies that reacted with other N- or C-terminal region p19 synthetic peptides. Polyclonal rabbit antisera to N- and C-terminal peptides reacted with a native viral protein of 19,000 daltons and with gag-encoded precursors of p19. Six monoclonal antibodies against native viral p19 were screened for reactivity to the five synthetic peptides. One of six antibodies (13B12) reacted with the C-terminal synthetic peptide of p19. Antibody 13B12 did not react with HTLVII or HTLVIII proteins or with HTLVIII-infected cells, nor did it cross-react with a wide variety of HTLV-uninfected normal host tissues. Thus, the C-terminus of p19 contains an antigen that is highly immunogenic in most HTLVI-infected patients and is HTLVI specific.
Antibodies, Monoclonal↗
Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV.
Approximately 80 percent of all human sera that react with antigens of HTLV-III, the etiologic agent of the acquired immune deficiency syndrome (AIDS), recognize protein bands at 66 and 51 kilodaltons. A mouse hybridoma was produced that was specific to these proteins. Repeated cloning of the hybridoma did not separate the two reactivities. The p66/p51 was purified from HTLV-III lysates by immunoaffinity chromatography and subjected to NH2-terminal Edman degradation. Single amino acid residues were obtained in 17 successive degradation cycles. The sequence determined was a perfect translation of the nucleotide sequence of a portion of the HTLV-III pol gene. The purified p66/51 had reverse transcriptase activity and the monoclonal immunoglobulin G specifically removed the enzyme activity from crude viral extract as well as purified enzyme.
Acquired Immunodeficiency Syndrome↗
Detection and characterization of the protein encoded by the v-rel oncogene.
To identify the protein encoded by v-rel, the oncogene of reticuloendotheliosis virus (REV-T), antisera have been raised to three synthetic peptides derived from the translation of our previously published v-rel DNA sequence [R.M. Stephens, N.R. Rice, R.R. Hiebsch, H.R. Bose, Jr., and R.V. Gilden, Proc. Natl. Acad. Sci. USA 80, 6229-6233 (1983)]. Sera to all three peptides precipitate a 59,000 Da protein from REV-T-transformed chicken lymphoid cells. This protein is not detectable in uninfected chick embryo fibroblasts, and its observed size is in good agreement with the 56,000 Da predicted by the DNA sequence. We conclude that this protein is the v-rel product and designate it p59rel. To search for evidence of post-translational processing of this protein, cells were grown in the presence of glycosylation inhibitors. These resulted in no detectable difference in the size of p59rel. Nor was its size detectably altered during the course of a pulse-chase experiment. Growth of cells in the presence of [32P] orthophosphate, however, revealed that p59rel is a phosphoprotein. It is also closely associated with a protein kinase activity, for precipitation with one of the peptide antisera (but not the other two) resulted in strong kinase activity in the immune complex pellet. During this reaction, p59rel itself becomes phosphorylated. Kinase activity was retained in the immune complex following detergent and high salt washes, leaving open the possibility that p59rel is itself a kinase.
Acetylglucosaminidase↗
Biosynthesis and chemical and immunological characterization of avian reticuloendotheliosis virus env gene-encoded proteins.
Two glycosylated proteins designated gp90 and gp20 were purified from replication-competent avian reticuloendotheliosis associated virus (REV-A). The N-terminal sequences of gp90 and gp20 were determined and found to match the REV-A-env-gene sequence. The alignments of the determined amino acid sequences with the predicted sequence indicate that gp20 and gp90 are the REV-A-encoded viral transmembrane and surface glycoprotein, respectively, and predict a signal peptide of 36 residues on the 5' end of the env-gene. Furthermore, gp90 of REV-A was detected by Western blot analysis with antibodies to a tridecapeptide corresponding to an env-gene nucleotide segment immediately preceding gp20 and thus representing the C-terminal portion of gp90. The env-gene precursor polyprotein gPr75-79env and Pr22(E), the precursor to gp20 and p2(E) were identified in the infected cells by monospecific antibodies raised against purified gp20. Thus the organization of gPR75-79env is likely to be N-gp90-gp20-p2(E), resembling that of M-MuLV gp85env. Sequence comparisons showed that the env gene of REV-A is highly related to both baboon endogenous virus and Type D retroviruses. In Western blot analyses, antibodies to REV-A gp20 cross-reacted with a panel of mammalian Type C and Type D viruses. Evolutionary aspects of these findings are discussed.
Amino Acid Sequence↗
Prospect for prevention of human immunodeficiency virus infection: purified 120-kDa envelope glycoprotein induces neutralizing antibody.
This study initiates an effort to develop a safe vaccine against the acquired immunodeficiency syndrome (AIDS) that is caused by infection with a retrovirus designated human immunodeficiency virus (HIV) [formerly human T-cell lymphotropic virus type III (HTLV-III)]. Other retrovirus models have shown that purified external glycoprotein subunits are immunogenic. The external envelope glycoprotein of HIV (gp120) has a molecular size of 120 kDa, is responsible for virus infectivity, and induces strong antibody response in humans. Purified HIV virus preparations contain relatively little gp120 so HIV-infected cells were used as the antigen source. The gp120 was localized on cell membranes and was solubilized with low levels of nonionic detergent. The glycoprotein was further purified by immunoaffinity chromatography over a resin prepared from IgGs isolated from patients. Homogeneity was achieved following extensive dialysis and polyacrylamide gel electrophoresis. The gp120 isolated from infected cells was shown to be structurally identical by peptide maps to virion gp120 and the amino-terminal amino acid sequence confirmed that the molecule was specified by the HIV genome. Goat, horse, and rhesus monkey (Macaca mulatta) immune sera to gp120 precipitated the homologous antigen and neutralized the in vitro infectivity of HIV. The induction of neutralizing antibody indicates that a gp120 subunit vaccine against HIV is theoretically possible.
Acquired Immunodeficiency Syndrome↗
Shedding and interspecies type sero-reactivity of the envelope glycopolypeptide gp120 of the human immunodeficiency virus.
Two glycopolypeptides with molecular weights 160,000 and 120,000 (gp120) are regularly recognized by human immunodeficiency virus (HIV)-specific antisera in lysates of cells persistently infected with HIV. In the present study, gp120 was characterized as the major envelope glycopolypeptide of HIV. Gp120 was identified as the external viral glycoprotein by radiosequencing and by its presence in purified virus. However gp120 was predominantly shed as a soluble protein into the culture fluid. Furthermore gp120 was precipitated by sera from horses infected with equine infectious anaemia virus (EIAV), but not by sera from uninfected animals. This may indicate conserved epitopes common to the envelopes of HIV and EIAV.
Antibodies, Viral↗
Bovine leukemia virus protease: purification, chemical analysis, and in vitro processing of gag precursor polyproteins.
Bovine leukemia virus protease was purified to homogeneity and assayed by using murine leukemia virus Pr65gag, a polyprotein precursor of the viral core structural proteins, as the substrate. A chemical analysis of the protease, including an amino acid composition and NH2- and COOH-terminal amino acid sequence analysis, revealed that it has an Mr of 14,000 and is encoded by a segment of the viral RNA located between the gag gene and the putative reverse transcriptase gene. As expected from the nucleotide sequence data (Rice et al., Virology 142:357-377, 1985), the reading frame for the protease is different from both the gag and reverse transcriptase reading frames. The 5' end of the protease open reading frame extends 38 codons upstream from the codon for the NH2-terminal residue of the mature viral protease and overlaps the gag open reading frame by 7 codons. The 3' end of the protease open reading frame extends 26 codons beyond the codon for the COOH-terminal residue of the mature protease and overlaps 8 codons of the reverse transcriptase open reading frame. Several lines of evidence, such as protein mapping of the gag polyprotein precursor, the characteristic structure of the mRNA, and promotion of the synthesis of a gag polyprotein precursor by lysine tRNA in vitro, suggest that the protease could be translated by frameshift suppression of the gag termination codon. In vitro synthesized bovine leukemia virus gag-related polyproteins were cleaved by the protease into fragments which were the same size as the known components of bovine leukemia virus, suggesting that the specificity of cleavage catalyzed in vitro by the purified protease is the same as the specificity of cleavage found in the virus.
Amino Acid Sequence↗
Isolation of a lentivirus from a macaque with lymphoma: comparison with HTLV-III/LAV and other lentiviruses.
A retrovirus has been isolated on the human T-cell line HuT 78 after cocultivation of a lymph node from a pig-tailed macaque (Macaca nemestrina) that had died with malignant lymphoma in 1982 at the University of Washington primate center. This isolate, designated MnIV (WPRC-1) (M. nemestrina immunodeficiency virus, Washington Primate Research Center) shows the characteristic morphology of a lentivirus and replicates to high titers in various lymphocyte lines of human and primate origin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified MnIV revealed multiple bands of structural proteins, including a major viral gag protein of 28 kilodaltons, that did not comigrate with the viral proteins of a human immunodeficiency virus (HIV [FRE-1]) that was also isolated on HuT 78 cells. The relatedness of MnIV to other lentiviruses (HTLV-III/LAV, EIAV, and visna) was examined in radioimmunoassays, by immunoblot techniques, and by N-terminal amino acid sequence analysis of the viral p28 gag protein. The immunoassays revealed cross-reactivity only between MnIV p28 and HTLV-III/LAV p24, and sequence analysis showed that 14 of the 24 N-terminal residues of MnIV p28 and HTLV-III/LAV p24 are identical. These results indicate that MnIV belongs to the same lentivirus family as HTLV-III/LAV but is only partially related to these human acquired immune deficiency syndrome retroviruses.
Amino Acid Sequence↗
Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins.
The retroviral transmembrane envelope protein p15E is immunosuppressive in that it inhibits immune responses of lymphocytes, monocytes, and macrophages. A region of p15E has been conserved among murine and feline retroviruses; a homologous region is also found in the transmembrane envelope proteins of the human retroviruses HTLV-I and HTLV-II and in a putative envelope protein encoded by an endogenous C-type human retroviral DNA. A peptide (CKS-17) was synthesized to correspond to this region of homology and was examined for its effects on lymphocyte proliferation. CKS-17 inhibited the proliferation of an interleukin-2-dependent murine cytotoxic T-cell line as well as alloantigen-stimulated proliferation of murine and human lymphocytes. Four other peptides, representing different regions of virus proteins, were inactive. These results suggest that the immunosuppressive portion of retroviral transmembrane envelope proteins may reside, at least in part, in a-conserved sequence represented by the CKS-17 peptide.
Amino Acid Sequence↗
Detection of the myristylated gag-raf transforming protein with raf-specific antipeptide sera.
The post-translational modifications of the gag-raf fusion proteins of the 3611 murine sarcoma virus (MSV) have been examined by inhibiting glycosylation with tunicamycin and by in vivo labeling with [3H]myristic acid. The results show that P75gag-raf is myristylated but not glycosylated and that P90gag-raf is glycosylated but not myristylated (and is now termed gP90gag-raf). gP90gag-raf expression appeared to become lost during passage of the transformed cells, and consequently does not appear to be necessary for the maintenance of transformation. raf-specific sera for detecting gag-raf fusion proteins have been obtained from synthetic peptides made from different regions of the predicted v-raf sequence. Immunoprecipitation of P75gag-raf with raf-specific sera directly confirmed the deduced v-raf sequence. The fact that P75gag-raf is both myristylated and precipitated by antiserum to a predicted carboxyl-terminal peptide of the v-raf gene established that the mature protein represents the entire coding region. The gP90gag-raf thus appears to be a glycosylated form of P75gag-raf specified by the gag sequences of the fusion protein, in analogy with Pr65gag and gPr80gag of murine leukemia viruses. Antiserum to the carboxyl-terminal P75gag-raf peptide was the most efficient in immunoprecipitation, and will be useful for detecting the product of the c-raf gene.
Animals↗
Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene.
Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.
Acquired Immunodeficiency Syndrome↗
Antibody to a synthetic peptide detects a conserved region of retrovirus transmembrane proteins.
N-terminal amino acid sequence analysis of the transmembrane protein of baboon endogenous virus revealed an internal 13 residue identity with the transmembrane homolog of murine leukemia virus. A tridecapeptide Glu-Val-Val-Leu-Gln-Asn-Arg-Arg-Gly-Leu-Asp-Leu-Leu corresponding to this region was chemically synthesized and antibody to the peptide was raised in rabbits. The rabbit antisera recognized the protein in Western blots. The specificity of the antisera was tested against a panel of retroviruses. Transmembrane proteins of type C retroviruses as well as type D were identified.
Amino Acid Sequence↗
Purification and chemical and immunological characterization of avian reticuloendotheliosis virus gag-gene-encoded structural proteins.
Five gag-gene-encoded structural proteins, designated p12, pp18, pp20, p30, and p10 were purified from replication-competent avian reticuloendotheliosis-associated virus (REV-A) by high-performance liquid chromatography complemented with chloroform-methanol extraction and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Based on amino acid composition and NH2- and COOH-terminal sequence analysis p12, pp18, p30, and p10 are distinct from one another, whereas pp20 is likely identical to pp18 in primary structure. The p12 was resistant to Edman degradation and was found to be myristylated at the NH2-terminal amino group. Sequence comparisons among the retrovirus family show that pp18/pp20 and p10 are, respectively, homologs of phospho-proteins and nucleic acid-binding proteins. A comparison of terminal sequences with the nucleotide sequence of spleen necrosis virus (SNV) revealed that the gag genes of SNV and REV-A are highly conserved; together with the identification of REV-A gag-precursor polyprotein, Pr60gag in immunoprecipitates of radiolabeled cell lysates, this comparison also led to the establishment of the organization of Pr60gag, viz., NH2-p12-pp18-p30-p10-OH. Sequence comparisons show that REV-A/SNV is related to mammalian type C viruses: the pp18-p30 region is most homologous to the macaque/colobus group and least to simian sarcoma virus (SSV), whereas both the 5'- and 3'-gag regions (i.e., p12 and p10) are clostest to SSV. Immunological studies using monospecific antisera and Western-blot analysis showed that antigenic determinants of REV-A p30 are conserved in most of mammalian type C and type D viruses, but those of REV-A p12 are shared only with simian sarcoma-associated virus (SSAV) and endogenous viruses of macaques.
Amino Acid Sequence↗
Primary structure and processing of gag and env gene products of human T-cell leukemia viruses HTLV-ICR and HTLV-IATK.
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Amino Acid Sequence↗
Amino- and carboxyl-terminal sequence of Moloney murine leukemia virus reverse transcriptase.
The NH2-terminal amino acid sequence of Moloney murine leukemia virus reverse transcriptase was determined to be Thr-Leu-Asn-Ile-Glu-Asp-Glu-Tyr-Arg-Leu-His-Glu-. The comparison of the amino acid analysis data obtained after carboxypeptidase Y digestion with the published nucleotide sequence (T. M. Shinnick, R. A. Lerner, and J. G. Sutcliffe, Nature (London) 293, 543-548, 1981) led to the conclusion that the COOH-terminus is Leu coded by CTC in nucleotide positions 4608-4610, and the tentative COOH-terminal sequence is Pro-Asp-Thr-Ser-Thr-Leu-Leu-OH. In light of these and previously reported results the complexity and map order of the pol gene are discussed.
Amino Acid Sequence↗
Murine leukemia virus protease is encoded by the gag-pol gene and is synthesized through suppression of an amber termination codon.
We have purified from Moloney murine leukemia virus (Mo-MuLV) a protease that has the capacity of accurately cleaving the polyprotein precursor Pr65gag into the mature viral structural proteins. Both the NH2- and COOH-terminal amino acid sequences have been determined and aligned with the amino acid sequence deduced from the DNA sequence of Mo-MuLV by other workers. The results show that: (i) the protease is located at the 5' end of the pol gene, and the first four amino acids are overlapped with the 3' end of the gag gene; (ii) the fifth amino acid residue is glutamine, which is inserted by suppression of the UAG termination codon at the gag-pol junction; and (iii) the protease is composed of 125 amino acids with calculated Mr = 13,315, and the COOH terminus of the protease is adjacent to the NH2 terminus of reverse transcriptase. The map order of the gag-pol gene is proposed to be 5'-p15-p12-p30-p10-protease-reverse transcriptase-endonuclease-3'.
Amino Acid Sequence↗
Translational readthrough of an amber termination codon during synthesis of feline leukemia virus protease.
Feline leukemia virus contains a protease which apparently has the same specificity as murine leukemia virus protease. It cleaves in vitro the Pr65gag of Gazdar-mouse sarcoma virus into the constituent p15, p12, p30, and p10 proteins. We purified the protease and determined its NH2-terminal amino acid sequence (the first 15 residues). Alignment of this amino acid sequence with the nucleotide sequence (I. Laprevotte, A. Hampe, C. H. Sherr, and F. Galibert, J. Virol. 50:884-894, 1984) reveals that the protease is a viral-coded enzyme and is located at the 5' end of the pol gene. As previously found for murine leukemia virus (Y. Yoshinaka, I. Katoh, T. D. Copeland, and S. Oroszlan, Proc. Natl. Acad. Sci. U.S.A. 82:1618-1622, 1985), feline leukemia virus protease is synthesized through in-frame suppression of the gag amber termination codon by insertion of a glutamine in the fifth position, and the first four amino acids are derived from the gag gene.
Amino Acid Sequence↗