55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.     

Primate retroviruses: immunological cross-reactivity between major structural proteins of new and old world primate virus isolates.

The major 35,000-molecular-weight internal antigen (p35) of the squirrel monkey retrovirus (SMRV) was isolated and partially characterized. Immunological analysis of SMRV p35 led to the demonstration of antigenic determinants common to SMRV and the Mason-Pfizer monkey virus (MPMV). A broadly reactive competition immunoassay was developed utilizing antiserum to MPMV to precipitate 125I-labeled SMRV p35. Although the major structural proteins of MPMV and SMRV competed with equal efficiency in this assay, type B and type C oncornavirus proteins lacked detectable reactivity. Antibodies reactive with the major structural proteins of both MPMV and SMRV were observed in sera of several normal rhesus monkeys with known prior exposure to MPMV-infected animals. These findings demonstrate the ability of sera from naturally immunized primates to recognize broadly reactive interspecies antigenic determinants shared by the major structural proteins of type D oncornaviruses, and they suggest possible horizontal transmission of MPMV among rhesus monkeys. Although sera from a number of squirrel monkeys contained antibody to SMRV p35, the possibility that this latter reactivity was due to endogenous virus activation rather than horizontal transmission cannot be ruled out.     

Isolation of a novel simian T-cell lymphotropic virus from Pan paniscus that is distantly related to the human T-cell leukemia/lymphotropic virus types I and II.

An unusual serological profile against human T-cell leukemia/lymphotropic virus type I and II (HTLV-I and -II) proteins was reported in several human Pygmy tribes in Zaire and Cameroon with serum antibodies reactive with gp21 and p24. Here we describe a similar pattern of serum antibodies in a colony of captive pygmy chimpanzees and the isolation of a novel retrovirus, simian T-cell lymphotropic virus from Pan paniscus (STLVpan-p), from the peripheral blood mononuclear cells of several seropositive animals. Cocultures of peripheral blood mononuclear cells from three seropositive pygmy chimpanzees with human cord blood mononuclear cells led to the expression of an HTLV-I- and HTLV-II-related virus initially demonstrated by electron microscopy. Furthermore, several of these cocultures became immortalized T-cell lines expressing the CD4+ CD8+ DR+ phenotype of mature activated T cells. Southern blotting and DNA sequencing of a PCR fragment of viral DNA from these cell cultures demonstrated a distant evolutionary relationship of these viruses to HTLV-I and -II and distinct from the known STLV isolates. We designated this virus STLVpan-p. A genealogical analysis of the captive pygmy chimpanzees colony, originated from wild-caught animals, revealed a prevalence of seropositive offspring from infected mothers, as also observed with HTLVs. The presence in this old African Great Ape species of a virus which is genetically quite distinct from HTLV-I and -II could provide new insights in the phylogenesis of STLVs and HTLVs and be instrumental in the discovery of related human viruses.     

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.     

Persistent human immunodeficiency virus type 1 infection of monoblastoid cells leads to accumulation of self-integrated viral DNA and to production of defective virions.

Cell-free virus preparations from persistently infected monoblastoid cells (HU937) become progressively less infectious during long-term passage. This effect is specific for cell lines derived from U937 and is not observed in persistently infected T-cell lines. Reduced infectivity is correlated with accumulation of unusual, high-molecular-weight, extrachromosomal forms of the human immunodeficiency virus type 1 (HIV-1) DNA. These DNA molecules contain multiple copies of the viral genome, and their structures are highly variable. Of 17 subclones of the HU937 cell line, 15 unique restriction fragment patterns were observed for the HIV-1 viral DNA. Structural analysis of these viral DNA species indicated that they were formed by sequential rounds of long terminal repeat-mediated integration of one circular DNA form into preexisting monomeric or multimeric structures. These viral DNA structures are termed nested self-integrates. Once formed, self-integrates prove to be stable and can be maintained for several months in culture. The unusual structures of HIV-1 DNA in persistently infected monoblastoid cells attest to an alternative to the accepted retrovirus life cycle. The self-integrated viral DNA species reported here may explain some aspects of the mechanism controlling establishment and maintenance of persistent HIV-1 infection in cells of the monocyte/macrophage lineage.     

Inhibition of virus production in peripheral blood mononuclear cells from human immunodeficiency virus (HIV) type 1-seropositive donors by treatment with recombinant HIV-like particles.

We have previously reported on the assembly of recombinant human immunodeficiency virus (HIV)-like particles that contain gag structural proteins and present env glycoproteins gp120 and gp41 on their surfaces (O. Haffar,. J. Garriques, B. Travis, P. Moran, J. Zarling, and S.-L. Hu, J. Virol. 64:2653-2659, 1990). On the basis of their structures, we hypothesized that the recombinant particles would interfere with virus infection and tested our hypothesis in vitro by using peripheral blood mononuclear cells (PBMC) from HIV type 1-seropositive donors. Addition of the recombinant particles to PBMC concomitant with stimulation by anti-CD3 inhibited virus production, as determined by reduced levels of p24 in the culture supernatants. This inhibition of p24 production correlated with lower levels of cell-associated viral DNA. Several lines of evidence suggested that the recombinant particles exerted their antiviral effects primarily by inhibiting virus production from latently infected cells and not by inhibiting subsequent virus spread. Importantly, CD4+ T-cell stimulation by specific antigen or by anti-CD3 was not inhibited by treatment with the recombinant particles. This apparent selective inhibition of virus replication in infected PBMC represents a novel property of the recombinant HIV-like particles.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

A mechanism of restricted human immunodeficiency virus type 1 expression in human glial cells.

We characterized in detail the life cycle of human immunodeficiency virus type 1 (HIV-1) in human glioma H4/CD4 cells which stably express transfected CD4 DNA (B. Volsky, K. Sakai, M. Reddy, and D. J. Volsky, Virology 186:303-308, 1992). Infection of cloned H4/CD4 cells with the N1T strain of cell-free HIV-1 (HIV-1/N1T) was rapid and highly productive as measured by the initial expression of viral DNA, RNA, and protein, but all viral products declined to low levels by 14 days after infection. Chronically infected, virus-producing H4/CD4 cells could be obtained by cell cloning, indicating that HIV-1 DNA can integrate and remain expressed in these cells. The HIV-1 produced in H4/CD4 cells was noninfectious to glial cells, but it could be transmitted with low efficiency to CEM cells. Examination of viral protein composition by immunoprecipitation with AIDS serum or anti-gp120 antibody revealed that HIV-1/N1T-infected H4/CD4 cells produced all major viral proteins including gp160, but not gp120. Deglycosylation experiments with three different glycosidases determined that the absence of gp120 was not due to aberrant glycosylation of gp160, indicating a defect in gp160 proteolytic processing. Similar results were obtained in acutely and chronically infected H4/CD4 cells. To determine the generality of this HIV-1 replication phenotype in H4/CD4 cells, nine different viral clones were tested for replication in H4/CD4 cells by transfection. Eight were transiently productive like N1T, but one clone, NL4-3, established a long-lived productive infection in H4/CD4 cells, produced infectious progeny virus, and produced both gp160 and gp120. We conclude that for most HIV-1 strains tested, HIV-1 infection of H4/CD4 is restricted to a single cycle because of the defective processing of gp160, resulting in the absence of gp120 on progeny 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.     

Different mechanisms account for the relative resistance of KG-1 and HL-60 cell lines to retrovirus infection.

I infected three different human leukemic cell lines (K562, KG-1, and HL-60) with an amphotropic retrovirus vector (designated PA317/N2) which confers G418 resistance and contains the Moloney murine leukemia virus long terminal repeat. Compared with K562 cells, both KG-1 and HL-60 cells were relatively resistant to infection with this retrovirus vector. In HL-60 cells, this resistance appeared to result from diminished viral DNA synthesis, while in KG-1 cells there was a block to the genomic integration of the viral DNA.     

A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.

A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.     

Cell-mediated immune response toward viral envelope and core antigens in gibbon apes (Hylobates lar) chronically infected with human immunodeficiency virus-1

The specific cellular immune response toward envelope and core proteins of human immunodeficiency virus-1 (HIV-1) was investigated in gibbon apes chronically infected with the HTLV-IIIB isolate. After in vitro stimulation of PBMC from infected and control animals with HIV-1 Ag, DNA synthesis, IL-2R expression and IL-2 release were assayed. Cells from infected gibbon apes demonstrated a group-specific response toward whole virus preparations from three divergent HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIMN). Consistent responses were also detected against purified HIV-1 Ag, i.e., native gp120 envelope glycoprotein, recombinant gp160 glycoprotein, a synthetic peptide (peptide 7) representing a highly conserved region of gp120, and purified native core protein p24. In addition, lymphocytes from infected gibbon apes displayed a specific, MHC-restricted, cytotoxic activity against autologous cells expressing HIV-1 envelope or gag proteins. The specific T cell reactivity toward HIV-1 proteins observed in infected gibbons contrasts with findings in HIV-1 infected humans, and may help to explain the apparent discrepancy in the natural history of the infection between the two species.     

Viral protein synthesis in mouse hepatitis virus strain A59-infected cells: effect of tunicamycin.

We identified eight protein species in virions of mouse hepatitis virus strain A59. Based on their sizes, prosthetic groups, and locations in virions, these proteins were designated gp180/E2, gp90/E2, pp54/N, gp26.5/E1, gp25.5/E1, p24/E1, p22/X, and p14.5/Y. The positions of the last two proteins in virions are not known. Host protein synthesis in Sac(-) cells infected with mouse hepatitis virus strain A59 was inhibited, and the following novel proteins appeared: gp150, gp90, p54, gp26.5, gp25.5, p24, p22, and p14.5. Except for gp150, these polypeptides all co-electrophoresed with mouse hepatitis virus strain A59 structural proteins. In addition, all of these proteins could be immunoprecipitated with a convalescent mouse serum or a rabbit antiserum raised against purified disrupted virus. After a 15-min pulse of infected cells with radioactive amino acids at 7h postinfection, gp90 was not detected, whereas gp26.5 and gp25.5 were only labeled to a small extent. During a subsequent chase period gp150 was processed to gp90, whereas the radioactivity in gp26.5 and gp25.5 increased concomitantly with a reduction of label in p24. Tunicamycin, an antibiotic which inhibits the synthesis of glycopeptides bearing N glycosidically linked oligosaccharides, prevented the appearance of gp150 in mouse hepatitis virus strain A59-infected cells. Instead, a 110,000-dalton protein accumulated. In contrast, the syntheses of the smaller viral glycoproteins gp26.5 and gp25.5 were resistant to this drug, indicating that these glycosylations were of the O glycosidical type. Although the production of infectious virus in tunicamycin-treated cells was inhibited by more than 99%, release of noninfectious viral particles continued. An analysis of these particles revealed that they lacked the peplomeric glycoproteins gp90/E2 and gp180/E2. Obviously, although the surface projections were not essential for budding of virus particles from the cells, they were required for infectivity.     

Radiommunoassays for the 70,000-molecular-weight glycoproteins of endogenous mouse type C viruses: viral antigen expression in normal mouse tissues and sera.

Genetic information coding for type C RNA viruses is transmitted within the DNA of mouse cells. At least three endogenous viruses have so far been immunologically distinguished by radioimmunoassays for their 12,000-molecular-weight polypeptides (p12). In the present study, the 70,000-molecular-weight glycoproteins (gp70) of three prototype viruses were purified, and competition radioimmunoassays were developed for each. By use of these immunoassays, the antigenic determinants of gp70's of different classes of endogenous virus, isolated from the same and from a variety of other mouse strains, were readily discriminated. In contrast, viruses of the same class were indistinguishable. These findings further document the existence of three distinct endogenous viruses of mouse cell. The levels of type C viral gp70 were quantitated in tissues and sera of several inbred strains. The pattern of immunological reactivity of the gp70 detected in serum was indistinguishable from that of the viral gp70 partially purified from tissues of the same strain. Moreover, in each case it was indistinguishable from that of a specific class of endogenous virus. In virus-negative tissues of BALB/c and NIH Swiss mice, the viral gp70 detected was shown to be representative of a class III endogenous virus whose p12 polypeptide was also expressed by the same cells.     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Primate retroviruses: intracistronic mapping of type D viral gag gene by use of nonconditional replication mutants.

Nonconditional replication mutants of squirrel monkey retrovirus (SMRV), an endogenous type D virus of primates, are shown to be defective in post-translational processing of nonglycosylated virus-coded structural proteins. Utilizing such mutants, in combination with sensitive radioimmunological assays, we demonstrate the existence of a 72,000-molecular-weight precursor polyprotein (Pr72gag) encoded by a region of the SMRV genome designated gag. Post-translational cleavage of this precursor polyprotein gives rise to virion structural proteins of 35,000 (p35), 16,000 (p16), 12,000 (p12), and 9,000 (p9) molecular weight. Three of these viral proteins, p35, p16, and p9, are shown to be phosphorylated. Analysis of viral antigen expression in cell lines nonproductively infected with either of two replication-defective SMRV mutants or mink cells productively infected with wild-type SMRV resulted in the detection of several SMRV Pr72gag intermediate cleavage products. Adjacent proteins within such intermediates are identified by use of specific competition immunoassays, and the intracistropic order of individual structural proteins with SMRV Pr72gag was tentatively deduced as NH2-p16-p12-p35-p9-COOH.