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PubMed1988

T-immunogenic peptides are constituted of rare sequence patterns. Use in the identification of T epitopes in the human immunodeficiency virus gag protein.

The sequences of a set of 63 peptides of demonstrated T immunogenicity have been analyzed and compared with two different randomly generated sets of sequences. This study indicates a statistically significant tendency of T immunogenic peptides to be constituted of clusters of rare tetrapeptides, as evaluated from the available sequence data banks. This result has been used to locate potential T epitopes in the human immunodeficiency virus (HIV) gag protein. Four peptides corresponding to the best candidate T epitopes (chosen in regions of conserved sequence among different virus isolates) have been synthesized and found to be recognized by a HIV-1-specific, HLA-A2-restricted human cytotoxic T cell line.

Amino Acid Sequence↗
PubMed1989

Detection of high concentrations of HTLV-1 p24 and a novel gag precursor, p45, in serum immune complexes of a healthy seropositive individual.

Circulating immune complexes from sera of 6 patients with adult T-cell leukemia and 8 asymptomatic carriers infected with the human T-cell leukemia virus type 1 (HTLV-1) were precipitated with polyethylene glycol, and the precipitates were tested for the presence of viral antigen p24 by Western blot, using affinity-purified radiolabelled goat antibody to p24 as a probe. p24 and a larger protein of Mr 45,000 immunologically related to p24 were detected at concentrations of up to 100-200 ng/ml in one of the carriers, while all other samples were negative. The concentration of the Mr 45,000 protein in the immune complexes was considerably higher than that of p24. Analysis of an HTLV-1 producer cell line, G-11/MJ, by competition Western blots and gag-specific antibodies, showed the presence of a hitherto unrecognized viral protein p45 which contains both p19 and p24 and probably represents an intermediate precursor of these proteins. The results present direct evidence of production of HTLV-1 proteins in an asymptomatically infected individual.

Antigen-Antibody Complex↗
PubMed1989

Recombinant HIV1 protease secreted by Saccharomyces cerevisiae correctly processes myristylated gag polyprotein.

The protease of the human immunodeficiency virus type I (HIV1) was expressed both intracellularly and extracellularly in Saccharomyces cerevisiae. Intracellular expression of the protease was achieved by fusing a 179 amino acid precursor form of the protease to human superoxide dismutase (hSOD). Self-processing of the viral enzyme from the hybrid precursor was demonstrated to occur within the yeast host. Secretion of the protease was achieved by fusing the leader sequence of yeast alpha-factor to the precursor form of the protease or to the 99 amino acid mature form of the protease. Authentic and active forms of the retroviral enzyme were detected in yeast supernatants of cells expressing the precursor or the mature form of the protease. A D25E active site variant of the retroviral enzyme exhibited diminished autocatalytic activity when expressed intracellularly or secreted from yeast. The wild-type protease was active in an in vitro assay on the natural substrate, myristylated gag precursor, Pr53gag. Correct processing of Pr53gag at the Tyr 138-Pro 139 junction was confirmed by amino terminal sequence analysis of the resulting capsid protein (CA, p24). The secreted protease was purified to homogeneity from yeast media using preparative isoelectric focusing and reverse-phase HPLC. Amino terminal sequence analysis showed a sequence beginning at amino acid 1 of the mature enzyme (Pro) and another sequence beginning at amino acid 6 (Trp). This shorter sequence may represent a natural autolytic product of the protease.

Amino Acid Sequence↗
PubMed1993

Methionyl-tRNA synthetase zinc binding domain. Three-dimensional structure and homology with rubredoxin and gag retroviral proteins.

Methionyl-tRNA synthetase from Escherichia coli contains one tightly bound zinc atom per subunit. The region encompassing residues 138 to 163 of this enzyme is responsible for the metal binding. A 28-mer peptide corresponding to these residues was expressed in vivo and shown to contain approximately 1 mol of tightly bound Zn/mol of peptide. In this study, the three-dimensional solution structure of this peptide was solved by means of two-dimensional proton NMR spectroscopy. A total of 133 nuclear Overhauser effect distance constraints and 22 dihedral angle restraints were used for the calculations, using a hybrid distance-geometry-simulated annealing strategy. Excluding the first four residues, the resulting structure is well-defined (r.m.s.d. 0.71 A for backbone atoms) and composed of a series of four tight turns. The second and the fourth turns are composed of CXXC sequences which are structurally homologous to the NH-S turns found in the metal binding sites of gag retroviral proteins and rubredoxin. The solution structure of the zinc binding peptide shows significant discrepancies with the crystal structure of methionyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1997

Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site.

Frameshift and readthrough sites within retroviral messenger RNAs are often followed by nucleotide sequences that have the potential to form pseudoknot structures. In the work presented here, NMR methods were used to characterize the base-pairings and structural features of the RNA pseudoknot downstream of the gag-pro frameshift site of simian retrovirus type-1 (SRV-1) and a functional mutant of the SRV-1 pseudoknot. Evidence is presented that these pseudoknots contain two A-form helical stems of six base-pairs each, connected by two loops, in a classic H-type pseudoknot topology. A particularly interesting feature is that the shorter of the two connecting loops, loop 1, consists of only a single adenosine nucleotide that spans the major groove of stem 2. In this respect, the frameshift-associated pseudoknots are structurally similar to the pseudoknot within the gene 32 mRNA of bacteriophage T2, previously characterized by NMR methods. Despite having similar nucleotide sequences, the solvent exchange rates of the imino protons at the junction of the helical stems in the wild-type and mutant frameshifting pseudoknots differ from each other and from the bacteriophage T2 pseudoknot. The implications of this finding are discussed.

Frameshifting, Ribosomal↗
PubMed1994

A murine leukemia virus expressed in aged DBA/2 mice is derived by recombination of the Emv-3 locus and another endogenous gag sequence.

Although most inbred strains of mice contain endogenous retroviral sequences, these sequences are usually not capable of producing infectious retroviruses. In some cases, the retroviral sequences are small fragments of viral genomes. In a few cases the sequences for complete retroviruses exist, but contain small defects which prevent the production of infectious virus. While the ability of reversions of these defects to produce retroviruses has been studied by site-directed mutagenesis and chemical-mediated mutagenesis, the presence of spontaneously occurring reversions has not been completely evaluated. We characterized an infectious ecotropic retrovirus spontaneously expressed in aged DBA/2 mice, which carries the complete but defective Emv-3. Although this endogenously produced retrovirus was related to the endogenous Emv-3 sequence, it had undergone recombination with another retroviral sequence to correct the defect which resided in the gag of Emv-3. Thus, recombination of endogenous ecotropic retroviral sequences may be a mechanism to produce infectious retroviruses in adult animals that contributes to pathologic disease.

3T3 Cells↗
PubMed2000

The kinetics of specific immune responses in rhesus monkeys inoculated with live recombinant BCG expressing SIV Gag, Pol, Env, and Nef proteins.

Development of an effective preventive or therapeutic vaccine against HIV-1 is an important goal in the fight against AIDS. Effective virus clearance and inhibition of spread to target organs depends principally on the cellular immune response. Therefore, a vaccine against HIV-1 should elicit virus-specific cytotoxic lymphocyte (CTL) responses to eliminate the virus during the cell-associated stages of its life cycle. The vaccine should also be capable of inducing immunity at the mucosal surfaces, the primary route of transmission. Recombinant Bacille Calmette-Guérin (BCG) expressing viral proteins offers an excellent candidate vaccine in view of its safety and ability to persist intracellularly, resulting in the induction of long-lasting immunity and stimulation of the cellular immune response. BCG can be administered orally to induce HIV-specific immunity at the mucosal surfaces. The immunogenicity of four recombinant BCG constructs expressing simian immunodeficiency virus (SIV) Gag, Pol, Env, and Nef proteins was tested in rhesus macaques. A single simultaneous inoculation of all four recombinants elicited SIV-specific IgA and IgG antibody, and cellular immune responses, including CTL and helper T cell proliferation. Our results demonstrate that BCG recombinant vectors can induce concomitant humoral and cellular immune responses to the major proteins of SIV.

Animals↗
PubMed1990

Substitutions at the P2' site of gag p17-p24 affect cleavage efficiency by HIV-1 protease.

Heptapeptide substrates containing a single amino acid substitution at the p2' position of the gag p17-p24 junction (S-Q-N-Y-P-X-V where X = G, A, L, I, F, and W) were compared as substrates for HIV-1 protease. Binding of the Ile-, Leu-, and Ala- containing peptides was about equal although hydrolysis was 20-fold lower for the Ala and Leu peptides compared to Ile. Insertion of Gly or Phe at the p2' position resulted in significantly lower cleavage of the peptide while a Trp-containing peptide was not cleaved. These data suggest that a relatively small hydrophobic amino acid is important for hydrolysis and binding at this site. Structure-activity studies such as those described here may be useful in the design of specific inhibitors for HIV-1 protease.

Amino Acid Sequence↗
PubMed1992

Production of a unique multi-lamella structure in the nuclei of yeast expressing Drosophila copia gag precursor.

Drosophila retrotransposon copia produces virus-like particles (VLPs) in the nuclei of cultured Drosophila cells. The VLPs contain copia RNA and reverse transcriptase activity, and thus, play a major role in copia replication. Here we have expressed the copia gag polyprotein precursor in yeast. The precursor, which includes copia protease itself, showed correct autoprocessing to produce a unique multi-lamella structure in the nuclei of the yeast cells. This expression system should be useful for the analysis of nuclear localization of the major copia VLP protein, and furthermore, would provide important information concerning the mechanism of copia VLPs formation.

Animals↗
PubMed1979

A clinical survey of gagging patients.

A clinical investigation was carried out on 74 dental patients who were suffering from a severe gagging reflex. The most common stimulating factor was the maxillary denture. Routine history and clinical examination were carried out for each patient, and lateral skull radiographs and an Eysenck Personality Inventory were completed for some of the patients. Treatment consisted of the use of an acrylic resin training base combined with relaxation therapy and heterohypnotic techniques. The results of this ongoing study were: (1) there were no consistent features of the group which differentiated them from control groups; (2) a number of patients were insufficiently motivated and discontinued treatment; and (3) some patients who were declared completely cured suffered relapses.

Adult↗
PubMed1992

Antibody titers to HTLV-I-p40tax protein and gag-env hybrid protein in HTLV-I-associated myelopathy/tropical spastic paraparesis: correlation with increased HTLV-I proviral DNA load.

We studied the relationship between antibody titers to recombinant HTLV-I p40tax protein and gag-env hybrid protein in serum (by an enzyme-linked immunosorbent assay) and HTLV-I proviral DNA load in peripheral blood mononuclear cells (by a quantitative polymerase chain reaction method) in 18 patients with HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), 17 HTLV-I carriers without HAM/TSP and 16 HTLV-I uninfected controls. The IgG and IgA antibody titers to either of the proteins correlated significantly with the HTLV-I pX (coding p40tax protein) and pol DNA amounts in HTLV-I infected subjects. HAM/TSP patients had significantly higher titers of IgG and IgA antibodies to the HTLV-I proteins than did the HTLV-I carriers without HAM/TSP. While the IgM antibodies to the HTLV-I proteins were found in only 6% of HTLV-I carriers without HAM/TSP, they were found in 40% of HAM/TSP patients, especially those having both a high HTLV-I proviral DNA load and high titers of the IgG and IgA antibodies. HAM/TSP patients with the IgM antibodies had a tendency to deteriorate more frequently on the Kurtzke's disability status scale and magnetic resonance imaging of the brain (leukoencephalopathy) than did those without in the two-year follow-up. Thus, the presence of IgM antibody and high titers of IgG and IgA antibodies to the HTLV-I proteins, together with the increased HTLV-I proviral DNA load, appears to distinguish HAM/TSP patients from HTLV-I carriers without HAM/TSP.

Adult↗
PubMed1984

Isolation and characterization of a Mo-MuSV-transformed TB cell line that produces noninfectious MuSV particles with uncleaved gag protein which is processed in the presence of Mo-MuLV.

A cell line, TBSV7, that produces noninfectious murine sarcoma virus (MuSV) in the absence of helper MuLV was isolated from TB cells infected with the supernatant of MuSV349 cells. These noninfectious MuSV particles with "immature" C-type virus morphology contain a 2.2 X 10(6)-Da genomic RNA and an uncleaved 62,000-Da gag precursor protein (Pr62). Neither viral envelope proteins (gp70, p15E, p12E) nor reverse transcriptase were detected in these virus particles. Pr62 was found to be phosphorylated in vivo and it could be phosphorylated in vitro with [gamma-32P]ATP, indicating that protein kinase was packaged in these noninfectious virions. In vitro processing of Pr62 to smaller molecular weight proteins could be achieved by the addition of Mo-MuLV and Nonidet P-40. The initial cleavage products were proteins with molecular weights of 38K (Pr38) and 27K (Pr27). Under optimum conditions Pr38 was cleaved to p30 and a protein band migrating with MuLV-p10, while Pr27 was cleaved to a 17,000-Da protein that migrated slower than MuLV-p15 and a protein band migrating with MuLV-p12. Pulse-chase experiments performed on TBSV7 cells superinfected with Mo-MuLV indicated that intracellular processing of Pr62 was much slower than that of Pr65. Cleavage protein products of Pr62 similar in size to the in vitro protein products were also detected in TBSV7 cells superinfected with MuLV.

Animals↗
PubMed1984

Immunological characterization of the low molecular weight gag gene proteins p19 and p15 of human T-cell leukemia-lymphoma virus (HTLV) and demonstration of human natural antibodies to them.

Small molecular weight gag gene proteins p19 and p15 of human T-cell leukemia virus (HTLV) were purified to homogeneity from density banded virus, using differential affinities to phosphocellulose at pH 7.9 and 6.5. Using specific radioimmunoassays, p19 and p15 were shown to band with HTLV on sucrose gradients. These proteins were present only in HTLV-I and in cell lines expressing HTLV-I. They were not expressed in a B-cell line from patient CR, although his T cells were HTLV producers. No significant immunological cross-reactivity was detected between these proteins and HTLV-IIMo. P19 and p15 did not share any common antigenic determinants with each other or with HTLV p24 in competition radioimmunoassays. Sera of HTLV-positive leukemia-lymphoma patients and some of their relatives also had natural antibodies to p19 and p15.

Antibodies, Viral↗
PubMed1984

Impaired cleavage of the joint gag-pol polyprotein precursor and virion assembly in a temperature-sensitive mutant of Rous sarcoma virus.

A temperature-sensitive coordinate mutant tsLA83 of Prague (PR-B) strain of Rous sarcoma virus at the nonpermissive temperature (41 degrees) produces noninfectious virus particles (NI-LA83) which contained only 3% of the reverse-transcriptase activity present in infectious virions. Analyses of [35S]methionine-labeled NI-LA83 showed the presence of all of the viral proteins except reverse transcriptase. Pulse-chase analyses of the virus-specified proteins in cells infected with LA83 or PR-B showed that the gag and glycoprotein precursors, Pr76gag and gPr95env, respectively, were processed at both 35 and 41 degrees. The reverse-transcriptase precursor, Pr180gag-pol, however, was not processed in LA83-infected cells at 41 degrees. In contrast, cells infected with LA83 or PR-B at 35 degrees as well as with PR-B at 41 degrees showed normal cleavage of Pr180gag-pol. A shiftdown of LA83-infected cells at 41 degrees to the permissive temperature 35 degrees resulted in the normal processing of Pr180gag-pol and production of infectious virus containing reverse transcriptase. Electron microscopic analysis showed that at 41 degrees cells infected with LA83 showed a large number of budding structures but fewer released particles. A shiftdown from 41 to 35 degrees resulted in an increase of virus particles with a concomitant decrease in budding structures suggesting that the processing of reverse-transcriptase precursor is related to virion assembly.

Animals↗
PubMed1985

Simian sarcoma virus-encoded gag-related protein: in vitro cleavage by Friend leukemia virus-associated proteolytic activity.

The simian sarcoma virus (SSV) encodes a gag-related 65,000-Da protein (SSV p65) which is not processed in SSV nonproducer cells (SSV-NP cells) (H.-J. Thiel, T. J. Matthews, E. M. Broughton, K. J. Weinhold, D. P. Bolognesi, T. Graf, and H. Beug (1981a), Virology 114, 124-131). In order to cleave SSV p65, retroviral particles containing this antigen were incubated with extracts from the heterologous helper virus Friend leukemia virus (FLV). Superinfection of SSV-NP cells by FLV has been previously shown to result in processing of SSV p65 in vivo (H.-J. Thiel, F. Weiland, R. Hafenrichter, T. J. Matthews, and K. J. Weinhold (1982), Virology 123, 229-234). In vitro cleavage was most efficient in the presence of a nonionic detergent (greater than 0.1% Nonidet-P40) and a reducing agent (greater than 5 mM dithiothreitol) at a pH of 7.0. The products, termed SSV p55 (p15, p12, p30), SSV p30, SSV p25 (p15, p12), and SSV p10, were characterized by (1) molecular weight, (2) kinetics experiments, (3) incorporation of different radiolabeled amino acids, and (4) comparison with SSAV structural proteins. Kinetics experiments with two amino acids ([3H]leucine, [35S]cysteine) revealed that initial processing of SSV p65 produced SSV p55 and SSV p10, with subsequent processing of SSV p55 occurring thereafter. In contrast to the Moloney system, the major intermediate p40 (p30, p10) could not be clearly demonstrated. A direct comparison of SSAV p10 and the cleavage product SSV p10 by SDS-PAGE suggests that SSAV pr65gag and SSV p65 differ slightly by molecular weight.

Animals↗
PubMed1986

The effect of cerulenin on the synthesis of the precursor gag polyprotein in defective murine leukemia and sarcoma virus producing cell lines.

The effect of cerulenin, an inhibitor of de novo fatty acid (and cholesterol) biosynthesis, on the synthesis of the precursor gag polyprotein, Pr65gag in a defective murine leukemia virus (334C) producing murine cell line (3JE) and a defective murine sarcoma virus (Gazdar) producing hamster cell line (HTG-2) was examined. In contrast to Moloney murine leukemia virus (M-MuLV) producing cell lines (MJD-54, clone 2) the amount of the Pr65gag remaining in the presence of cerulenin (20 micrograms/ml) was greatly reduced in both defective virus-infected cells. This effect appears specific for the Pr65gag polyprotein, since the env precursor polyprotein Pr80env was normally synthesized and remained undegraded in cerulenin-treated 3JE-infected cells. Thin-section electron micrographs showed an increased accumulation of virion particles in vesicles of treated HTG-2 cells.

Antifungal Agents↗
PubMed1987

Characterization of a virus-specific proteolytic activity processing the gag precursor of the simian sarcoma-associated virus.

The proteolytic processing of the gag precursor polypeptide pr65gag of simian sarcoma-associated virus (SSAV) has been studied in vivo and in vitro. In SSAV-infected cells (i.e., in vivo) proteins of 52 and 38 kDa and the viral protein p30 could be immunoprecipitated with anti-p30 serum. This cleavage pattern is only in part imitated by in vitro cleavage of the isolated pr65gag with avian myeloblastosis virus (AMV) protease p15. However, in vitro incubation of isolated pr65gag with detergent-disrupted SSAV particles generated products identical in size to those found in vivo, i.e., proteins of 52 and 38 kDa and p30. The extent of cleavage is dependent on the concentration of the disrupted virions added to the incubation mixture. Studies with protease inhibitors suggest that the SSAV enzyme is a serine-type protease like that of other mammalian retroviruses and unlike the protease of avian viruses. The SSAV protease activity eluted from a molecular sieve column in a range of about 10-15 kDa reflecting the molecular weight of the murine leukemia virus (MuLV) protease (Mr = 13.5K). Thus, it appears that there is a close similarity between the proteolytic enzymes present in different mammalian retroviruses such as MuLV and SSAV.

Avian Myeloblastosis Virus↗
PubMed1990

Evidence that HIV-1 gag precursor shares antigenic sites with the major capsid protein of human cytomegalovirus.

A rabbit antiserum prepared against disrupted sucrose-banded HIV-1 virus (strain FRE-3) reacted with antigens present in nuclear inclusions, pathognomonic for human cytomegalovirus (HCMV). This cross-reactivity was observed in autopsy specimens from individuals infected with CMV, in the presence or absence of co-infection with HIV-1. A Towbin immunoassay showed that the serum reacted specifically with the HCMV major capsid protein (MCP, 153 kDa), both in the nuclear fraction of infected cells and in virions. Direct evidence that these proteins share antigenic determinants was provided by the two-way cross-reactivity of affinity-selected antibodies (i.e., anti-MCP with HIV-1 gag precursor Pr55; anti-Pr55 with MCP). All four strains of HCMV tested showed this reactivity, but the counterpart proteins of simian CMV and herpes simplex virus type 1 did not, indicating that the determinant is not common to all herpes group viruses.

Antigens, Viral↗