Human immunodeficiency virus type 1 tropism for T-lymphoid cell lines: role of the V3 loop and C4 envelope determinants.
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Amino Acid Sequence↗
Biomedical subjects
L Ratner
Publications and source records attributed to L Ratner.
Cooperative effects of the human immunodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells.
Insertion of T-cell line-tropic V3 and V4 loops from the HXB2 strain into the macrophage-tropic YU-2 envelope resulted in a virus with delayed infectivity for HUT78 and Jurkat cells compared with HXB2. Sequence analysis of viral DNA derived from long-term cultures of Jurkat cells revealed a specific mutation that changed a highly conserved Asn residue in the V1 loop of Env to an Asp residue (N-136-->D). Introduction of this mutation into clones containing a T-cell line-tropic V3 loop, either with or without a T-cell line-tropic V4 loop, resulted in viruses that replicated to high levels in Jurkat cells and peripheral blood lymphocytes. The Env proteins from these constructs were expressed with the vaccinia virus/T7 hybrid system and were found to be translated, processed, and cleaved and to bind to soluble CD4 similar to the wild-type HXB2 and YU-2 Env proteins. Env-mediated fusion with HeLa T4+ cells, however, was regulated by both the altered V1 loop and T-cell line-tropic V3 loop. These results suggest that subsequent to the initial gp120-CD4 binding event, a functional interaction can occur between the altered V1 loop and T-cell line-tropic V3 loop that results in infection of Jurkat cells and peripheral blood lymphocytes.
Amino Acid Sequence↗
Weekly oral etoposide in patients with Kaposi's sarcoma associated with human immunodeficiency virus infection: a phase I multicenter trial of the AIDS Clinical Trials Group.
We conducted a Phase I trial to evaluate the safety, maximally tolerated dose (MTD), antitumor activity, and pharmacology of once-weekly oral etoposide in patients with Kaposi's sarcoma (KS) and AIDS. From September 1990 to October 1991, 27 eligible patients with biopsy-confirmed KS were treated at six etoposide dose levels, ranging from 150 to 400 mg weekly. Patients were treated until their tumor progressed or until unacceptable toxicity developed. On the first day of therapy, etoposide plasma concentrations were measured by high-performance liquid chromatography. The MTD was defined as the etoposide dose that induced reversible grade 3 toxicity in three of six patients during the first 4 weeks. Although dose-limiting toxicity was uncommon during the first 4 weeks of treatment (three of 27 patients), and the MTD was not reached, with longer treatment > 50% of patients developed dose-limiting toxicities, most commonly neutropenia. Responses were observed at all dosage levels (except 350 mg weekly), with partial tumor regression documented in nine (36%) of 25 evaluable patients. There was marked variability in etoposide area under the plasma concentration versus time curve, elimination half-time (t1/2), and urinary excretion. These pharmacokinetic features were not, however, associated with the presence of gastrointestinal symptoms, the severity of side effects, or tumor response. We conclude that weekly oral etoposide can be safely administered to patients with AIDS and KS. The observed antitumor effects over a wide range of doses support further studies with very low and minimally toxic etoposide doses, alone or in combination with other agents.
Acquired Immunodeficiency Syndrome↗
HIV-1 Nef protein downregulation of CD4 surface expression: relevance of the lck binding domain of CD4.
HIV Nef protein downregulates the surface expression of CD4 on T-cells but its mechanism of activity is unknown. We have analyzed the relevance of different portions of the CD4 molecule with respect to the activity of Nef. Upon transfection of Jurkat T-cells that express Nef or do not express Nef with constructs that include different domains of the CD4 molecule, we demonstrated that Nef downregulation of CD4 requires the first 30 amino acids of the CD4 cytoplasmic tail and that the lck-binding domain of CD4 was at least partially responsible for this effect. Furthermore, upon transfection of U-937 cells with an lck-expressing plasmid we showed that CD4 downregulation by Nef is significantly more efficient when lck is present. Finally, by measuring the rate of CD4 endocytosis by a novel flow cytometric method we showed that the effect of Nef on CD4 surface expression resulted from accelerated CD4 internalization.
Amino Acid Sequence↗
Development of leukemia in mice transgenic for the tax gene of human T-cell leukemia virus type I.
The human T-cell leukemia virus type I Tax protein trans-activates several cellular genes implicated in T-cell replication and activation. To investigate its leukemogenic potential, Tax was targeted to the mature T-lymphocyte compartment in transgenic mice by using the human granzyme B promoter. These mice developed large granular lymphocytic leukemia, demonstrating that expression of Tax in the lymphocyte compartment is sufficient for the development of leukemia. Furthermore, these observations suggest that human T-cell leukemia virus infection may be involved in the development of large granular lymphocytic leukemia.
Animals↗
Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA.
The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.
Adult↗
A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles.
Incorporation of Vpr into human immunodeficiency virus type 1 (HIV-1) virions is mediated by the Gag protein, independently of other viral components. We have coexpressed Vpr and Gag constructs in a vaccinia virus expression system in order to map the region of Gag involved in Vpr packaging. Deletion of the carboxyl-terminal p6 region of Gag impaired the ability of Gag to package Vpr. To confirm the role of p6 in Vpr packaging, Rous sarcoma virus (RSV)-HIV chimeras containing HIV-1 p6 were constructed. Although RSV Gag does not package Vpr into virus particles, a chimera containing HIV-1 p6 is sufficient for Vpr incorporation. To map the region of p6 involved in Vpr packaging, a series of p6 point mutations and deletion mutations was analyzed. Mutations in the N-terminal p6 proline-rich domain, for which preliminary evidence shows a marked decrease in virion incorporated RNA, did not affect Vpr incorporation. Deletion of residues 1 to 31 of HIV-1 p6 did not affect Vpr packaging, but residues 35 to 47, including an (LXX)4 domain, were required for Vpr incorporation into virus particles.
Amino Acid Sequence↗
HIV-1 V3 envelope sequences required for macrophage infection.
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Amino Acid Sequence↗
Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones.
Molecular clones of human T-cell leukemia virus type 1 (HTLV-1) have been constructed and stably propagated in plasmids in Escherichia coli. Expression of Tax could be demonstrated from these clones in fibroblast and epithelial cell lines. HOS cells stably transfected with HTLV-1 clone ACH produced all three classes of viral transcripts and Gag proteins. Virus-like particles were also produced from ACH transfected HOS cells as demonstrated by sucrose gradient and electron microscopic analyses. Transfection of peripheral blood mononuclear cells with ACH resulted in production of infectious virus particles which induced lymphocyte proliferation. This study describes useful reagents for further examination of the biological properties of HTLV-1.
Base Sequence↗
Killing of primary CD4+ T cells by non-syncytium-inducing macrophage-tropic human immunodeficiency virus type 1.
Understanding the mechanism by which human immunodeficiency virus type 1 (HIV-1) kills CD4+ T lymphocytes is important to the development of therapeutic and prophylactic strategies. Recent studies have indicated that, in some cases, progression to AIDS is associated with the appearance of syncytium-inducing, T cell line-tropic HIV-1 variants. Nevertheless, approximately 50% of subjects with AIDS harbor only non-syncytium-inducing, macrophage-tropic (NSI-M) variants of HIV-1. In most asymptomatic patients, NSI-M HIV-1 isolates are the predominant virus type found. We report here that cytopathicity of NSI-M HIV-1 for primary CD4+ T lymphocytes can be directly detected in vitro. The extent of CD4+ T-cell killing was not completely correlated with the rate of viral replication, suggesting that other characteristics of HIV-1 contribute to its cytopathicity. Our findings suggest that: (i) direct killing by NSI-M HIV-1 may contribute to CD4+ T-lymphocyte depletion in vivo, and (ii) the determinants of HIV-1 cytopathicity for CD4+ T lymphocytes and cell tropism or syncytia-forming ability are not necessarily tightly linked.
Acquired Immunodeficiency Syndrome↗
The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells.
The replication of human immunodeficiency virus type 1 (HIV-1) in nondividing host cells such as those of macrophage lineage is an important feature of AIDS pathogenesis. The pattern of HIV-1 replication is dictated, in part, by the nucleophilic property of the viral gag matrix (MA) protein, a component of the viral preintegration complex that facilitates nuclear localization of viral nucleic acids in the absence of mitosis. We now identify the accessory viral protein Vpr, as a second nucleophilic component that influences nuclear localization of viral nucleic acids in nondividing cells. Reverse transcription and nuclear localization of viral nucleic acids following infection of cells by viruses lacking Vpr or viruses containing mutations in a gag MA nuclear localization sequence were indistinguishable from the pattern observed in cells infected by wild-type HIV-1. These viruses retained the ability to replicate in both dividing and nondividing host cells including monocyte-derived macrophages. In contrast, introduction of both gag MA and Vpr mutations in HIV-1 attenuated nuclear localization of viral nucleic acids in nondividing cells and virus replication in monocyte-derived macrophages. These studies demonstrate redundant nucleophilic determinants of HIV-1 that independently permit nuclear localization of viral nucleic acids and virus replication in nondividing cells such as monocyte-derived macrophages. In addition, these studies provide a defined function for an accessory gene product of HIV-1.
Base Sequence↗
Regulation of human immunodeficiency virus Nef protein by phosphorylation.
Human immunodeficiency virus isolates express a Nef protein with either an alanine or a threonine at amino acid residue 15. The threonine residue is a site for phosphorylation by protein kinase C. Jurkat T cells constitutively expressing the alanine variant of Nef exhibit the ability to downregulate the induction of transcription factors NF-kB and AP-1. In contrast, Jurkat cells with the threonine variant of Nef are at least partially restored in their ability to recruit NF-kB and AP-1.
Binding Sites↗
Structure and expression of the human T-cell leukemia virus type 1 envelope protein.
The structure and expression of the HTLV-1 envelope protein was examined using T lymphoid cell lines infected with HTLV-1 and recombinant vaccinia viruses expressing the HTLV-1 envelope. Pulse-chase experiments demonstrated that the envelope precursor, gp62, had a half-life of 7-12 hr. N-glycosylation of the precursor protein was examined using tunicamycin and endoglycosidase H. These studies revealed that at least four and possibly five potential N-glycosylation sites were utilized. In addition, the envelope precursor was found to sediment on sucrose gradients as high-molecular-weight complexes, in positions consistent with the formation of dimers and smaller amounts of higher multimeric forms. Finally, the recombinant vaccinia system was used to express mutants designed to analyze the role in HTLV-1 envelope processing of the cytoplasmic tail and the membrane-spanning domain.
Base Sequence↗
HIV-2 viral protein X association with the GAG p27 capsid protein.
VPX is a 16 kDa accessory protein expressed in cells infected with HIV-2 and most SIV strains and is packaged into virus particles. In order to define the requirements for incorporation of VPX into virions, VPX and HIV-2 GAG-POL were expressed independently from a vaccinia virus-based transient expression system. Under these conditions, VPX was exported from transfected cells only when coexpressed with the HIV-2 GAG-POL plasmid. A 27 kDa protein coprecipitating with VPX was found to have an identical electrophoretic mobility as the GAG capsid protein, and reacted with an anti-GAG antiserum. Coexpression of VPX and GAG-POL resulted in virus-like particles containing both proteins, as determined by sucrose gradient analyses. Expression of VPX and HIV-2 GAG without POL gave similar results. VPX association with HIV-2 GAG p27 capsid protein was specific, since no association was found with the HIV-1 GAG p25/p24 capsid protein.
Animals↗
Particle assembly and Vpr expression in human immunodeficiency virus type 1-infected cells demonstrated by immunoelectron microscopy.
The 96 amino acid viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) was detected during virus assembly in intracellular vacuoles and at the plasma membrane on peripheral blood mononuclear cells. In both immature and mature virus particles, Vpr was located immediately beneath the viral envelope, colocalizing with the core structural protein, Gag p24. Vpr was present in intracellular HIV-1 wild-type virions at 50% of the level found in extracellular HIV-1 particles. Cells infected with HIV-1 strains with C-terminal truncations of Vpr manifested a different pattern of Vpr expression. A mutant with an alteration of amino acids 79 to 85 exhibited a 23% reduction in total levels of Vpr expression, but a marked accumulation of Vpr in intracellular rather than extracellular virions. A mutant with the last 17 amino acids of Vpr deleted expressed only 10% of wild-type levels of Vpr. These observations indicate that Vpr is incorporated into virions from the cytoplasmic aspect of either the vacuolar or plasma membrane. Furthermore, the proportion of Vpr on intracellular compared to extracellular virions is affected by a specific locus within the protein.
Amino Acid Sequence↗
CD3-dependent lymphocyte activation by human T cell leukaemia virus type I-producing T cells.
Human T cell leukaemia virus type I-(HTLV-I) transformed cells are capable of stimulating the proliferation of normal T lymphocytes, or stimulating interleukin 2 expression in Jurkat T lymphoid cells. This effect is mediated by the CD2/lymphocyte function-associated antigen 3 (LFA-3) adhesion/signalling pathway. The current work demonstrates that CD3 is also required for this effect, suggesting that the T cell receptor (TCR)/CD3 complex is mediating this effect. However, this effect does not appear to be due to a superantigen since no change in TCR expression was found after HTLV-I-mediated proliferation, nor was proliferation inhibited by an antibody against the specific TCR expressed on Jurkat cells (TCR V beta 8).
Adult↗
Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.
Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.
Animals↗