SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Explore the source record for details and available documents.
Cell lines stably releasing noninfectious virus-like particles containing wild type or mutant gene products represent useful tools for a biochemical, immunological, and structural analysis of virus assembly. Human immunodeficiency virus (HIV) type 1 gag and env gene products were transiently and stably expressed in mammalian cells and the formation of virus-like particles incorporating viral glycoproteins was analyzed. Transient cotransfection of plasmids directing the synthesis of gag and env gene products yielded efficient release of particles but specific incorporation of HIV glycoproteins was not detected. A stable cell line expressing wild type HIV-1 glycoproteins was generated and transient transfection of this cell line with gag-encoding constructs led to the release of virus-like particles incorporating HIV surface and transmembrane glycoproteins. Attempts to establish stable cell lines expressing wild type HIV gag and pol genes were unsuccessful and only highly unstable lines primarily expressing uncleaved precursor polyproteins were obtained. This result appears to be caused by the cytotoxic effects of the viral proteinase since stable lines were readily selected after transfection of constructs either encoding an inactive mutant of the proteinase or a mutated frameshift signal which prevented expression of the pol reading frame. Stable coexpression of uncleaved Gag polyprotein and wild type env gene products yielded efficient release of immature virus-like particles incorporating HIV glycoproteins. Electron micrographs revealed lentiviral budding structures with the typical surface projections of viral glycoprotein oligomers.
The products of the env gene of HIV-1 play critical roles in determining the host range of the virus and it cytopathicity, as well as forming an important focus of an infected individual's immune response to the virus. We describe the isolation and characterization of separate HeLa cell lines expressing the env gene products of three highly divergent strains of HIV-1.
The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.
The gag and env gene products of human T-cell leukemia virus (HTLV) were identified with rabbit antisera against the synthetic peptides and a polypeptide produced in Escherichia coli, which corresponded to parts of the proteins predicted from the nucleotide sequence of HTLV [M. Seiki, S. Hattori, Y. Hirayama, and M. Yoshida (1983). Proc. Natl. Acad. Sci. USA 80, 3618-3622]. Viral proteins were detected by immunoprecipitation in two HTLV-producing cell lines. The precursor of gag products was a protein with an apparent molecular weight of 53,000 (Pr53), and was shown to be processed into three mature gag proteins, p19, p24, and p15, in this order, from the 5' end of the gag gene. The processing sites were confirmed to be the same as those predicted from the nucleotide sequence. The env gene product was identified as a glycoprotein of 62,000 Da (gp62), which was processed into gp46 and p20E. All the viral antigens described above were also detected with sera from ATL patients, indicating that all these proteins are expressed in the patients.
The structural requirements for proteolytic cleavage of the human immunodeficiency virus type 1 env gene product, gp160, to gp120 and gp41 have been assessed by specific mutagenesis of the sequence Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg located between amino acids 500 and 511, i.e., at the putative C terminus of gp120. The basic amino acids underlined have been mutated, individually and in combination, to neutral amino acids, and the cleavability of the mutated env gene products was examined after expression in CV-1 cells. The results show that the replacement of Arg-511 (cleavage presumably occurs C terminal to this amino acid) with Ser completely abolishes recognition and cleavage by the cellular protease(s), i.e., the remaining basic amino acids in the vicinity do not serve as alternative substrates. However, Arg-508 and Lys-510 are important features of the recognition site since, when they are individually changed to neutral amino acids, cleavage is severely impaired. The basic amino acids 500, 502, and 504 are, individually, not important for cleavage, since their individual replacement by neutral amino acids does not impair cleavage. However, when all four basic amino acids 500, 502, 503, and 504 are changed to neutral amino acids, cleavage is almost completely abolished. This shows that the sequence Arg Glu Lys Arg at the cleavage site is alone not sufficient for cleavage but that a contribution of other amino acids is required, whether the other amino acids provide a basic character or a certain structure in the vicinity of the cleavage site. When noncleavable or poorly cleavable mutant env genes are expressed from the infectious plasmid pNL4-3 in CD4+ human lymphoblastoid cells, noninfectious virus, incapable of spread throughout the culture, is produced.
Five in-frame stop mutations in the HIV-1 env gene, which lead to the production of env gene products truncated within the cytoplasmic C-terminal tail, have been generated and their effects on membrane fusion capacity, glycoprotein incorporation into virus particles, infectivity, and cytopathogenicity were analyzed. The resulting truncated glycoproteins were processed normally, were transported to the cell surface, and were able to induce CD4-dependent membrane fusion. The membrane fusion capacity of one of the mutant glycoproteins with a truncation of 144 amino acids was increased to about double of that induced by wild-type glycoprotein. With a single exception, the truncated viral glycoproteins were incorporated into virus particles which were infectious and cytopathic for permissive MT-4 cells. The infection kinetics with the mutated viruses were, however, delayed to varying degrees in comparison to infection with wild-type virus. Nevertheless, in each case, PCR amplification and direct sequencing of viral DNA in the infected cultures confirmed the presence of the mutant and the absence of revertant DNA. The mutant virus encoding a viral glycoprotein with the longest truncation (144 amino acids), in which only 7 cytoplasmic C-terminal amino acids in gp41 remain, resulted in infection kinetics in MT-4 cells which were only marginally delayed in comparison to those induced by wild-type virus. This means that these C-terminal 144 amino acids of gp41 are not necessary for glycoprotein incorporation into virus particles nor do they significantly contribute to the infectivity nor the cytopathogenicity of HIV-1 in MT-4 cells.
We have vaccinated five groups of cats (n = 25) four times with five preparations of recombinant feline immunodeficiency virus (FIV) env gene products; one group (n = 7) served as control. The vaccine formulations were as follows: (1) envelope glycoprotein of FIV Zurich 2 (FIV Z2) expressed in a Baculovirus system and isolated by gel electroelution (denatured form); (2) insect cells expressing FIV Z2 glycoprotein; (3) envelope glycoprotein of a Boston strain (FIV Bangston) expressed in insect cells and isolated by gel electroelution (denatured form); (4) glycosylated Bangston envelope protein made in insect cells and isolated in a native form; (5) non-glycosylated Bangston envelope protein made in Escherichia coli. All cats were challenged with 20 50% cat infective doses (CID50) of FIV Z2 previously titrated in cats. All vaccinated cats developed high enzyme-linked immunosorbent assay (ELISA) antibodies to the homologous antigen; crossreactivity to heterologous antigens was seen at a lower level. Virus neutralizing antibodies (tested with Petaluma virus) reached titers up to 32. After challenge, all cats seroconverted (as judged by anti gag antibodies in Western blot) and became infected (as judged by virus isolation and/or polymerase chain reaction) between 4 and 11 weeks with the exception of one cat. It is concluded that it is relatively easy to induce high ELISA antibody titers using recombinant env gene products, ELISA antibody titers do not correlate with virus neutralization or with protection.
The mature envelope glycoproteins of mouse mammary tumor virus (gp52 and gp36) were isolated by reversed-phase high-pressure liquid chromatography. The N-terminal amino acid sequence of gp36 was determined for 28 residues. The C-terminal amino acid sequences of gp52 and gp36 were determined by carboxypeptidase digestion. The N-terminal amino acid sequence of gp52 has been reported previously (L. O. Arthur et al., J. Virol. 41:414-422, 1982). These data were aligned with the predicted amino acid sequence of the env gene product obtained by translation of the DNA sequence (S. M. S. Redmond and C. Dickson, Eur. Mol. Biol. Org. J. 2:125-131, 1983). The amino acid sequences of the mature viral proteins were in agreement with the predicted amino acid sequence of the env gene product over the regions of alignment. This alignment showed the sites of proteolytic cleavages of the env gene product leading to the mature viral envelope glycoproteins. The N-terminal amino acid sequence of gp52 starts at residue 99 of the predicted structure indicating proteolytic cleavage of a signal peptide. A dipeptide (Lys-Arg) is excised between the C-terminus of gp52 and the N-terminus of gp36. The C-terminal amino acid sequence of gp36 is identical to the sequence predicted by the codons immediately preceding the termination codon for the env gene product. The data show that there is no proteolytic processing at the C-terminal of the murine mammary tumor virus env gene product and that the env gene coding region extends into the long terminal repeat.
A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.
Polyadenylylated mRNA isolated from cells infected with Rauscher murine leukemia virus was fractionated by centrifugation in in a denaturing sucrose gradient into different sizes. Each RNA fraction was injected into oocytes of Xenopus laevis and the virus-specific products were analyzed by immunoprecipitation with polyvalent and monospecific antisera against polypeptides of Rauscher murine leukemia virus, and then by gel electrophoresis and scintillation autoradiography. It was shown that a 35S mRNA species directs the synthesis of a precursor of the internal or group-specific antigens of the virion (the gag-gene products). A 22S mRNA species directs the synthesis of two viral envelope polypeptides and their precursor polypeptide (env-gene products). The results indicate that the gag- and env-related polypeptides of Rauscher murine leukemia virus are synthesized uncoordinately and provide evidence for open and closed cistrons on the virus-specific mRNAs.
Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, encodes two glycosylated virion proteins, gp20 and gp70. The polyprotein precursor to these proteins was identified by immunoprecipitation of pulse labeled M-PMV-infected cells with an antiserum raised against gp70, the major glycoprotein of the virus. The relationship of this precursor to the two viral glycoproteins was verified by tryptic peptide mapping, which demonstrated that gp20 and gp70 were independent products of the env gene. The types and degree of glycosylation of the precursor and its products was investigated by tunicamycin inhibition of glycosylation, endo-beta-N-acetyl glucosaminidase H (Endo-H) and endo-beta-N-acetylglucosaminidase F (Endo-F) catalyzed removal of glycosylated residues. The results suggest that the precursor, a molecule with a mol wt of 86,000, is composed of approximately 55,000 Da of protein to which 14-15 oligosaccharide chains are attached. The precursor is cleaved post-translationally to yield the two glycoproteins of M-PMV, gp70 and gp20. Most, if not all, of the glycan units associated with the gp70 molecule are of the complex variety, as shown by their resistance to Endo-H cleavage. The gp20 molecule, on the other hand, appears to contain a single glycan unit predominantly of the high mannose type since this side chain is sensitive to digestion by Endo-H.
Protection against human T-cell leukemia virus type-I (HTLV-I) infection in cynomolgus monkeys, achieved by immunizing the animals with env gene products of HTLV-I produced in Escherichia coli, was evaluated. Four monkeys that had been immunized with the env product produced antibody against HTLV-I gp68 and gp46, and their sera were found to cause strong inhibition of syncytium formation of a cat fibroblast cell line induced by HTLV-I. Immunized and non-immunized monkeys were challenged with live MT-2 cells, a high HTLV-I-producer cell line. After challenge, all the control non-immunized monkeys were infected with HTLV-I, as judged by the frequent detection of HTLV-I-antigens in cultures of their peripheral blood mononuclear cells (PBMC), whereas no antigens were recovered from PBMC of immunized monkeys. These results indicate that humoral immunity against HTLV-I-envelope protein elicited by immunization with the polypeptides synthesized in bacteria protected the monkeys against primary infection with HTLV-I.
Two inhibitors of glycosylation, 2-deoxyglucose and tunicamycin, depressed the synthesis of infectious Rous sarcoma virus greater than 100-fold. Under the same conditions only a two- to threefold decrease in the production of virus particles was observed. The noninfectious particles had a lower density (1.145 g/ml) in isopycnic sucrose gradients and lacked the two virion glycoproteins, gp85 and gp37, found on infectious virions. The four internal structural proteins of the virus, p27, p19, p15, and p12, appeared to be assembled normally into the noninfectious virus. Polypeptides related to the Rous sarcoma virus glycoproteins were immunoprecipitated from pulse-labeled Rous sarcoma virus (Prague strain, subgroup B)-transformed cells. pr95gp, the polyprotein precursor to gp85 and gp37, was the major protein precipitated from untreated cells. PR95GP, THE POLYPROTEIN PRECURSOR TO GP85 AND GP37, WAS THE MAJOR PROTEIN PRECIPITATED FROM UNTREATED CELLS. This was absent in both tunicamycin- and 2-deoxyglucose-treatec ells, and a new polypeptide of molecular weight 57,000 to 58,000 was the major species precipitated. In tunicamycin-treated cells this product was unstable and was degraded during a 2-h chase; in 2-deoxyglucose-treated cells, on the other hand, the polypeptide appeared to be more stable and underwent partial glycosylation. The synthesis and processing of pr76, the polyprotein precursor to the internal structural proteins of the virion, occurred normally in both treated and untreated cells. It is concluded that the unglycosylated env gene product is a polypeptide of molecular weight 57,000 to 58,000.
Expression of DNA sequences homologous to sequences of env gene of mouse mammary tumor virus (MMTV) in the lymphocytes of patients with breast cancer and in subjects at a high risk of breast cancer has been reported. Antigen analogous to envelope protein gp52, product of MMTV env gene, is detected in T lymphocytes of virtually all patients with breast cancer and extremely rarely in T cells of controls, where its expression is confined to B cells. For explaining such unexpected results, we studied the molecular basis of this antigen synthesis. Specific PCR products were obtained using primers to gp52-coding region of MMTV env gene. One of them (957 nucleotides) was used as a probe for hybridization of DNA and RNA from lymphocytes of patients with breast cancer and controls. This sequence was hybridized with 90% frequency with genome DNA of breast cancer patients and with 85% frequency with genome RNA of such patients, which is almost 4-fold more than in the controls (patients with gynecological tumors or donors). These results correlate with the frequency of detection of the studied antigen in patients with breast cancer and control group patients.
In the present study, mice each given a single intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) responded with increased serum levels of the major envelope glycoprotein, gp70, of endogenous retrovirus. Concentrations of gp70 in their sera began to increase 4 hr after LPS injection, reached maximal 5- to 15-fold increases after 12--24 hr, and returned to the preinjection levels within 3 days. This response occurred only in the strains characterized by high base line levels of serum gp70 (greater than 10 micrograms/ml) such as NZB, NZB X NZW F1, BXSB, MRL, NZW, DBA/2, LG, 129(GIX+), and C57BL/6(GIX+). However, strains such as DBA/1, C3H/St, BALB/c, C57BL/6(GIX-), and 129(GIX-) with lower base line levels of serum gp70 (less than 5 micrograms/ml) made little or no response. This serum gp70 induced by LPS was structurally similar to the gp70 of NZB xenotropic virus that is dominantly expressed in sera from virtually all strains of mice. However, (i) the induced gp70 was virion-free; (ii) xenotropic virus was not isolatable from BXSB, MRL/1, or 129(GIX+) mice injected with LPS; and (iii) amounts of the major structural viral protein, p30, did not increase correspondingly in sera. All of these findings indicate that the increased expression of serum xenotropic viral gp70 in response to LPS did not result from activation of replication-competent xenotropic virus. In addition, the serum gp70 response to LPS was abolished by simultaneous inoculation of an inhibitor of protein synthesis, D-galactosamine. These results strongly suggest that LPS selectively stimulates synthesis of the env gene product, gp70, of NZB xenotropic virus but other viral gene products.
Mouse 3T6 cells infected with Murine Leukemia Virus (MuLV) were cloned to yield several sublines producing viruses distinct from one another with respect to the ratio of uncleaved to cleaved gag gene-coded polyprotein, Pr65gag. The virus produced by the cloned sublines also differed in the ratio of the env gene-coded protein, p15E, to its product, p12E. The two ratios, Pr65gag/p30 and p15E/p12E, were found to be highly correlated among the cloned cell lines. Velocity gradient separation of the virions produced by individual sublines, followed by polypeptide analysis, demonstrated that the particles were inhomogeneous with respect to extent of cleavage both of PR65gag and of p15E. The two cleavages were again highly correlated. These data indicate that the gag and env gene product cleavages are not independent events but are tightly coupled.
The H strain of avian erythroblastosis virus (AEV-H) was recently isolated from the liver emulsion of a chicken that suffered from erythroblastosis after being inoculated with subgroup A leukosis virus. AEV-H induced erythroblastosis or sarcoma when inoculated into chickens and transformed chick embryo fibroblasts (CEF) in vitro. Analysis of viral proteins synthesized in cells, which were named HNP, transformed by AEV-H but not producing transforming virus revealed tha the genome of AEV-H directed the synthesis of the gag gene products, Pr76gag and Pr180gag-pol, which was the precursor of active reverse transcriptase. Thus the HNP produced virions that were not infectious due to a defect of the env gene. Studies on the viral RNA showed that a 35 S RNA, estimated to be 8000 nucleotides long, was the genomic RNA of AEV-H and probably carried one transforming gene, which is most likely erbB gene. The gene organization of AEV-H was suggested to be 5'-gag-pol-onc-3'. These data imply that the single oncogene is responsible for both erythroblastosis and sarcoma.