Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.     

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.     

Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule

Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.     

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.     

Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.     

Serologic identification and characterization of a macaque T-lymphotropic retrovirus closely related to HTLV-III

Human T-lymphotropic virus type III (HTLV-III) is thought to play an etiologic role in the development of the acquired immune deficiency syndrome (AIDS). In this study the serologic characterization of a new simian retrovirus that is related to HTLV-III is described. This new virus, here referred to as STLV-III, was isolated from sick macaques at the New England Regional Primate Research Center. Radioimmunoprecipitation analysis revealed STLV-III-specific proteins of 160, 120, 55, and 24 kilodaltons, all similar in size to the major gag and env proteins of HTLV-III. These antigens were recognized by representative macaque serum samples and human reference serum samples positive for HTLV-III antibodies. Monoclonal antibodies directed to p24, the major core protein of HTLV-III, also immunoprecipitated a 24-kilodalton species in lysates of cells infected with the macaque virus. This HTLV-III-related virus, which naturally infects a nonhuman primate species, may provide a useful model for the study of HTLV-III and the pathogenesis of AIDS.     

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.     

Genomic diversity of human T-lymphotropic virus type III (HTLV-III)

The DNA genomes of human T-lymphotropic virus type III (HTLV-III) isolated from 18 individuals with AIDS or who were at risk for AIDS were evaluated for evidence of variation. Although all of the 18 viral DNA's hybridized throughout their entire genomes to a full-length cloned probe of the original HTLV-III isolate, each of the 18 isolates showed a different restriction enzyme pattern. The number of restriction site differences between isolates ranged from only 1 site in 23 to at least 16 sites in 31. No particular viral genotype was associated with a particular disease state and 2 of the 18 patients had evidence of concurrent infection by more than one viral genotype. Propagation of three different viral isolates in vitro for up to 9 months did not lead to detectable changes in their restriction patterns. These findings indicate that different isolates of HTLV-III comprise a spectrum of highly related but distinguishable viruses and have important implications regarding the pathogenicity of HTLV-III and attempts to develop effective diagnostic, therapeutic, and preventive measures for this virus.     

A cytoplasmic protein inhibits the GTPase activity of H-Ras in a phospholipid-dependent manner

A cytoplasmic protein has been identified that inhibits the guanosine triphosphatase (GTPase) activity of bacterially synthesized, cellular H-Ras protein. This GTPase inhibiting protein is able to counteract the activity of GTPase activating protein (GAP), which has been postulated to function as a negative regulator of Ras activity. The potential biological importance of the GTPase inhibiting protein is further supported by its interaction with lipids. Phospholipids produced in cells as a consequence of mitogenic stimulation increase the activity of the GTPase inhibiting protein, as well as inhibit the activity of GAP. The interaction of such lipids with each of these two regulatory proteins would, therefore, tend to increase the biological activity of Ras and stimulate cell proliferation.     

Molecular cloning and expression of neuroleukin, a neurotrophic factor for spinal and sensory neurons

A novel 56,000-dalton growth factor found in mouse salivary gland was purified, molecularly cloned, and expressed in monkey COS cells. The protein is a neurotrophic factor and also, surprisingly, a lymphokine product of lectin-stimulated T cells. The factor was therefore named neuroleukin. Neuroleukin promotes the survival in culture of a subpopulation of embryonic spinal neurons that probably includes skeletal motor neurons. Neuroleukin also supports the survival of cultured sensory neurons that are insensitive to nerve growth factor, but has no effect on sympathetic or parasympathetic neurons. The amino acid sequence of neuroleukin is partly homologous to a highly conserved region of the external envelope protein of HTLV-III/LAV, the retrovirus that causes acquired immune deficiency syndrome.     

A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients

A newly identified protein from HTLV-III/LAV, the virus implicated as the etiologic agent of the acquired immune deficiency syndrome, was studied. This protein, which has a molecular weight of 27,000 (p27), was shown by amino acid sequencing to have a coding origin 3' to the env gene on the HTLV-III genome. The presence of antibodies to p27 in virus-exposed individuals indicated that this gene is functional in the natural host.     

施肥对火棘淀粉和可溶性蛋白含量影响的研究

[目的]为火棘果的进一步开发提供理论依据。[方法]对人工种植的火棘施纯氮0(N0)、23 g/株(N23),P2O5 0(P0)、23 g/株(P23),K2O0(K0)、23 g/株(K23),研究N、P、K对火棘淀粉和可溶性蛋白含量的影响。[结果]不同施肥处理火棘淀粉和可溶性蛋白含量有差异。N0P23K0处理的淀粉含量最高,最低的是N23P23K0;N23P0K0处理的可溶性蛋白含量最高,最低的是N23P23K23。N23P23K0、N0P23K23和N23P0K0处理的蛋白图谱中比其他处理多了一条分子量为51.766 kD的蛋白带。火棘可溶性蛋白的分子量集中在50～63 kD,属于低分子量蛋白。[结论]N、P、K相互作用对火棘淀粉的合成与积累有一定的作用,对蛋白的合成有较大的影响。     

Evidence for exposure to HTLV-III in Uganda before 1973

Fifty of 75 serum samples collected in the West Nile district of Uganda between August 1972 and July 1973 contained antibodies reactive with human T-cell leukemia (lymphotropic) virus type 3 (HTLV-III; mean titer, 601), while 12 of 75 samples were positive in a similar test for HTLV type 1 (HTLV-1) antibodies (mean titer, 236). The samples were screened by enzyme-linked immunosorbent assay and positive results were confirmed by a newly developed unlabeled antibody-peroxidase procedure with enhanced sensitivity for detection of antibody binding to immunoblots of HTLV-III antigen, demonstrating antibodies to proteins with molecular weights of 24,000, 41,000, and 76,000 in nearly all positive samples. Analysis of titration data indicated enhanced titers of antibody against HTLV-III and HTLV-I when coinfection occurred. The high prevalence and relatively low titers [compared to serum from patients with acquired immune deficiency syndrome (AIDS)] of antibodies recognizing HTLV-III proteins in sera from this population at a time that may predate or coincide with the appearance or spread of the AIDS agent (HTLV-III) suggest that the virus detected may have been a predecessor of HTLV-III or is HTLV-III itself but existing in a population acclimated to its presence. It further suggests an African origin of HTLV-III.     

HTLV-III in the semen and blood of a healthy homosexual man

Human T-lymphotropic virus type III (HTLV-III) is the probable etiologic agent for the acquired immune deficiency syndrome (AIDS). HTLV-III was isolated from semen and blood of a healthy homosexual man whose serum contains antibodies to HTLV-III. The finding of virus in semen supports epidemiologic data that suggest that AIDS can be transmitted sexually. In addition, the demonstration of HTLV-III in the blood and semen of a healthy individual establishes an asymptomatic, virus-positive carrier state which may be important in the dissemination of HTLV-III and, consequently, AIDS.     

Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome

The human T-cell leukemia (lymphotropic) virus type III (HTLV-III) appears to be central to the causation of the acquired immune deficiency syndrome (AIDS). Two full-length integrated proviral DNA forms of HTLV-III have now been cloned and analyzed, and DNA sequences of the virus in cell lines and fresh tissues from patients with AIDS or AIDS-related complex (ARC) have been characterized. The results revealed that (i) HTLV-III is an exogenous human retrovirus, approximately 10 kilobases in length, that lacks nucleic acid sequences derived from normal human DNA; (ii) HTLV-III, unlike HTLV types I and II, shows substantial diversity in its genomic restriction enzyme cleavage pattern; (iii) HTLV-III persists in substantial amounts in cells as unintegrated linear DNA, an uncommon property that has been linked to the cytopathic effects of certain animal retroviruses; and (iv) HTLV-III viral DNA can be detected in low levels in fresh (primary) lymphoid tissue of a minority of patients with AIDS or ARC but appears not to be present in Kaposi's sarcoma tissue. These findings have important implications concerning the biological properties of HTLV-III and the pathophysiology of AIDS and Kaposi's sarcoma.     

Expression and characterization of the trans-activator of HTLV-III/LAV virus

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.