Biomedical subjects

E Terwilliger

Publications and source records attributed to E Terwilliger.

14 recordsLinked to original sources

PubMed1997

Identification of HIV-1 determinants for replication in vivo.

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

Amino Acid Sequence↗

PubMed1996

Cyclic AMP-mediated growth arrest is associated with increased expression of human T cell leukemia virus type I structural and transforming genes.

The effect of increased intracellular cyclic AMP levels on gene expression of the human T cell leukemia virus type I (HTLV-I) provirus was examined. Induction of infected cells to produce elevated levels of cyclic AMP was associated with specific increases in viral surface antigen expression, protein synthesis, p24 release into the supernatant, and RNA levels. The patterns of HTLV-I proviral gene expression observed support results from transfection experiments regarding the function of Tax, Rex, and cyclic AMP in HTLV-I gene regulation. As evidenced by thymidine incorporation, treatment of the infected cells to produce cyclic AMP caused reversible growth arrest. The data indicate that HTLV-I RNA and protein synthesis can proceed at an elevated level in the absence of cell growth. Sustained increases in the intracellular level of cyclic AMP may represent a method for enriching cell cultures in HTLV-I proteins.

Bucladesine↗

PubMed1993

Changes in the host range and growth potential of an HIV-1 clone are conferred by the vpu gene.

vpu, alone among the genes of human immunodeficiency virus type 1 (HIV-1), is unique to this virus, with no analogous reading frame evident in the genomes of either HIV-2 or the related SIVs. Effects of vpu upon levels of virus production from infected cells have been noted, but vpu is dispensable for HIV-1 replication in vitro and its significance in the natural viral life cycle is unclear. We have now identified and characterized a major influence of vpu upon the growth and host cell range of a cloned recombinant HIV-1. This effect required the presence of a second determinant mapping to the 3' end of the env gene, and was not detected in clones incorporating an env gene derived from a different strain of the virus. This indicates that important effects of vpu may have been lost in some experimental systems because of the particular viruses used, and further suggests the involvement of a determinant in the transmembrane glycoprotein of the virus in the action of vpu.

Cell Line↗

PubMed1992

Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell lines. The infectivity of vif-defective viruses depended on the cell type in which the virus was produced. In CEM cells, vif was required for production of virus capable of initiating infection in all cell lines studied. vif-defective virus produced by SupT1, C8166, and Jurkat cells and the monkey cell line COS-1 could initiate infection in multiple cell lines, including CEM and H9. These results suggest that vif can compensate for cellular factors required for production of infectious virus particles that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in others such as CEM and H9 as well as peripheral blood T lymphocytes. The effect of vif was not altered by deletion of the carboxyl terminus of gp41, a proposed target for vif (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). These studies demonstrate that vif enhances viral infectivity during virus production and also suggest that vif is likely to be important for natural infections.

Amino Acid Sequence↗

PubMed1992

Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins.

The role of the cytoplasmic domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in virus replication was investigated. Deletion of residues 840 to 856 at the carboxyl terminus of gp41 reduced the efficiency of virus entry during an early step in the virus life cycle between CD4 binding and formation of the DNA provirus without affecting envelope glycoprotein synthesis, processing, or syncytium-forming ability. Deletion of residues amino terminal to residue 846 was associated with decreased stability of envelope glycoproteins made in COS-1 cells, but this phenotype was cell type dependent. The cytoplasmic domain of gp41 was not required for the incorporation of the HIV-1 envelope glycoproteins into virions. These results suggest that the carboxyl terminus of the gp41 cytoplasmic domain plays a role in HIV-1 entry other than receptor binding or membrane fusion. The cytoplasmic domain of gp41 also affects the stability of the envelope glycoprotein in some cell types.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus.

The art gene product of the human immunodeficiency virus is required for the expression of virion capsid and envelope gene proteins. The experiments presented here show that sequences located within the coding region of the envelope gene exert a negative effect on the expression of heterologous genes and that the negative effect of these sequences can be relieved by the art gene product. This region in the env gene contains negative regulatory sequences that inhibit gene expression, as well as a sequence necessary for the art gene product-dependent relief of repression. The experiments define the cis- and trans-acting components of a regulatory system that permits differential expression of human immunodeficiency virus virion structural and regulatory proteins.

Capsid↗

PubMed1988

The art gene product of human immunodeficiency virus is required for replication.

A mutation that alters the initiation codon of the art gene of the human immunodeficiency virus renders the virus replication defective. The replication defect of this mutation can be complemented in trans by the art gene product. These findings indicate that the art protein is essential for replication and, as such, represents a good target for development of new antiviral agents that could be useful in the control of human immunodeficiency virus infection.

Cells, Cultured↗

PubMed1988

Cell lines that express stably env gene products from three strains of HIV-1.

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.

Antibodies, Viral↗

PubMed1987

Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1.

The envelope of the human immunodeficiency virus type 1 (HIV-1) plays a central role in the process of virus entry into the host cell and in the cytopathicity of the virus for lymphocytes bearing the CD4 molecule. Mutations that affect the ability of the envelope glycoprotein to form syncytia in CD4+ cells can be divided into five groups: those that decrease the binding of the envelope protein to the CD4 molecule, those that prevent a post-binding fusion reaction, those that disrupt the anchorage of the envelope glycoprotein in the membrane, those that affect the association of the two subunits of the envelope glycoprotein, and those that affect post-translational proteolytic processing of the envelope precursor protein. These findings provide a functional model of the HIV envelope glycoprotein.

Amino Acid Sequence↗

PubMed1986

Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA.

We have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind 125I-labeled EGF; each lacks a functional EGF receptor. We used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in 125I-labeled cell extracts or in [35S]methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

Animals↗

PubMed1986

Effects of mutations within the 3' orf open reading frame region of human T-cell lymphotropic virus type III (HTLV-III/LAV) on replication and cytopathogenicity.

Mutations that eliminated the ability of the human T-cell lymphotropic virus type III to produce the 27-kilodalton 3' orf product did not eliminate the ability of the virus to replicate in and kill T4+ cells. A mutant carrying a mutation that deleted carboxy-terminal sequences from the envelope gene as well as the 3' orf sequences retained the ability to kill T4+ lymphocytes, but had a retarded replication rate.

Cell Line↗

PubMed1985

Dominant and recessive mitogen-nonproliferative variants of 3T3 cells.

We have previously isolated 3T3 cell variants unable to respond to specific mitogens. In this report we analyze the dominant and/or recessive nature of these variants. Two independently isolated EGF nonproliferative variants are unable to bind EGF. Hybrids between 3T3R5 cells (thymidine kinase deficient, ouabain-resistant) and these variants express EGF receptors; the "EGF receptorless" phenotype of these variants is recessive. Hybrids between these two variants do not bind EGF; they are defective in a common, non-complementing function. A TPA nonproliferative 3T3 variant is also recessive; hybrids with 3T3R5 mount a mitogenic response to TPA. In contrast a fourth variant, which can neither bind labeled EGF nor respond to TPA, is dominant for both characteristics. Hybrids between this latter variant and 3T3R5 can neither bind EGF nor mount a mitogenic response to TPA.

Animals↗

PubMed1984

3T3 variants unable to bind epidermal growth factor cannot complement in co-culture.

A Swiss-Webster 3T3 variant, called 3T3-ENR7, unable to divide in response to epidermal growth factor was isolated by the mitogen-colchicine selection technique. Like the other EGF non-proliferative variants (3T3-NR6 and 3T3-TNR2), 3T3-ENR7 was unable to bind 125I-EGF. Pairwise co-culture of the three independently isolated EGF non-responsive variants did not restore mitogenic responsiveness to EGF.

Animals↗

PubMedDate not supplied

A second post-transcriptional trans-activator gene required for HTLV-III replication.

Evidence is provided for the existence of a seventh gene in the genome of human T-lymphotropic virus type III/lymphadenopathy-associated virus. The gene is necessary for replication and acts post-transcriptionally to relieve negative regulation of the messenger RNA for the virion capsid and envelope proteins. These observations suggest mechanisms for regulating both the latent and lytic phases of the virus life cycle.

Cell Line↗