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M J Potash

Publications and source records attributed to M J Potash.

At least 19 recordsLinked to original sources
PubMed1997

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.

Escherichia coli↗
PubMed1997

Isolation and long-term culture of primary ocular human immunodeficiency virus type 1 isolates in primary astrocytes.

Vitreous specimens from 14 HIV-1 infected persons undergoing medically indicated vitrectomy were assayed for the presence of infectious HIV-1 and viral tropism. Human primary fetal astrocytes, adult lymphocytes, or macrophages were exposed to vitreous in culture and and cells were then assayed for HIV-1 DNA by polymerase chain reaction amplification. We found that 11 of 14 patients tested carried ocular HIV-1 which replicated in one or more primary cell types; of the 13 vitreous samples tested in astrocytes, eight contained transmissible HIV-1. The three patients with no culturable ocular virus were in antiviral therapy at the time of vitrectomy. Comparison of envelope V3 sequences from astrocytes infected in culture to that in uncultured blood cells revealed 21% sequence divergence indicating that ocular HIV-1 transmitted to astrocytes was not recently derived from virus present in the blood. Two ocular samples transmissible to astrocytes were tested further and found capable of sustained replication by serial passage to uninfected astrocytes. However, the viral structural proteins produced by infected astrocytes were abnormal, p24 was absent and higher molecular weight Gag proteins were present. We conclude that the eye is a central nervous system compartment which frequently contains HIV-1 capable of replication in human astrocytes.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Interference to human immunodeficiency virus type 1 infection in the absence of downmodulation of the principal virus receptor, CD4.

It is thought that interference during human immunodeficiency virus type 1 (HIV-1) infection is established by downmodulation of the principal virus receptor, CD4. Here we present evidence to the contrary. At various times after primary infection, we superinfected T cells in vitro by exposure to a genetically distinct viral clone or to a virus carrying the chloramphenicol acetyltransferase gene. Replication of each virus strain was determined by restriction enzyme analysis of total cellular DNA, by PCR amplification of viral DNA, or by assay of cell extracts for chloramphenicol acetyltransferase activity. We found that efficient viral interference is established within 24 h of infection at a multiplicity of infection of 1. At that time, expression of viral structural proteins was low and infected cells displayed undiminished levels of surface CD4 and were fully susceptible to virus binding and fusion. Superinfection by either cell-free HIV-1 or cocultivation was blocked. Cells resistant to superinfection by HIV-1 remained susceptible to Moloney murine leukemia and vaccinia viruses. No interference was observed 4 h after primary infection or in cells infected with either UV-inactivated HIV-1 or a mutant virus defective in virus-cell fusion activity, indicating that binding of primary virus to CD4 is insufficient to prevent superinfection. The minimum viral requirements for this interference are that HIV-1 must be able to enter cells and synthesize viral DNA; Tat-mediated transcription is dispensable. Our results support the existence of a novel pathway to interference to HIV-1 infection, which we term postentry interference, which blocks superinfection during intracellular phases of the virus life cycle.

Base Sequence↗
PubMed1996

vif-negative human immunodeficiency virus type 1 persistently replicates in primary macrophages, producing attenuated progeny virus.

The vif gene of human immunodeficiency virus type 1 (HIV-1) is required for efficient infection of primary T lymphocytes. In this study, we investigated in detail the role of vif in productive infection of primary monocyte-derived macrophages (MDM). Viruses carrying missense or deletion mutations in vif were constructed on the background of the monocytotropic recombinant NLHXADA-GP. Using MDM from multiple donors, we found that vif mutants produced in complementing or partially complementing cell lines were approximately 10% as infectious as wild-type virus when assayed for incomplete, complete, and circularized viral DNA molecules by quantitative PCR amplification or for viral core antigen p24 production by enzyme-linked immunosorbent assay. We then determined the structure and infectivity of vif mutant HIV-1 by using MDM exclusively both for virus production and as targets for infection. Biosynthetic labeling and immunoprecipitation analysis of sucrose cushion-purified vif-negative HIV-1 made in MDM revealed that the virus had reduced p24 content compared with wild-type HIV-1. Cell-free MDM-derived vif mutant HIV-1 was infectious in macrophages as determined by the synthesis and maintenance of full-length viral DNA and by the produc- tion of particle-associated viral RNA, but its infectivity was approximately 2,500-fold lower than that of wild-type virus whose titer was determined in parallel by measurement of the viral DNA burden. MDM infected with MDM-derived vif-negative HIV-1 were able to transmit the virus to uninfected MDM by cocultivation, confirming the infectiousness of this virus. We conclude that mutations in vif significantly reduce but do not eliminate the capacity of HIV-1 to replicate and produce infectious progeny virus in primary human macrophages.

Base Sequence↗
PubMed1995

Redefinition of tropism of common macrophage-tropic human immunodeficiency virus type 1.

Previous studies have suggested that the abilities of human immunodeficiency virus type 1 (HIV-1) to infect primary macrophages and transformed T cell lines are mutually exclusive and define an important biological distinction among HIV-1 strains. In a survey of eight macrophage-tropic HIV-1 strains and nine T cell lines, all frequently used in studies of tropism, we have found that six virus strains replicate in one or more T cell lines and that four T cell lines are highly susceptible to macrophage-tropic HIV-1. Passage through T cell lines did not affect the tropism or the env V3 sequence of monocytotropic HIV-1 strains. We conclude that HIV-1 replication in transformed T cells and primary macrophages are not mutually exclusive, and that as such, these definitions of tropism per se are not generally useful markers for other biological properties of HIV-1.

Amino Acid Sequence↗
PubMed1994

Modulation of cellular gene expression of HIV type 1 infection as determined by subtractive hybridization cloning: downregulation of thymosin beta 4 in vitro and in vivo.

Chronic infection with HIV-1 has profound effects on host cell growth and function. We used subtractive hybridization cloning to identify genes whose expression is modulated by HIV-1 infection in the T leukemia cell line CEM. The gene encoding thymosin beta 4, a ubiquitous polypeptide associated with hematopoietic differentiation, showed two- to threefold reduced transcription in HIV-1-infected CEM cells and other HIV-1-infected T cells and macrophages in vitro. Solid-phase radioimmunoassay revealed about a threefold decrease in the level of thymosin beta 4 protein in lysates of infected cells. Northern blot analysis of RNA samples from lymphocytes of five AIDS patients reveals an up to fivefold reduction in the level of thymosin beta 4 mRNA. These results indicate that HIV-1 infection may directly influence the expression of certain physiologically important proteins.

Cell Line↗
PubMed1993

Inhibition of type 1 human immunodeficiency virus replication by a tat antagonist to which the virus remains sensitive after prolonged exposure in vitro.

The transactivator of transcription, Tat, of human immunodeficiency virus type 1 (HIV-1) is required for viral replication. Inhibition of Tat function could have the potential to keep integrated provirus in dormancy. In the presence of Tat, Ro 24-7429, an analog of Ro 5-3335, inhibited expression of indicator genes controlled by the HIV-1 long terminal repeat promoter in transient transfection assays and in a constitutive cell line at noncytotoxic concentrations. Reduction of steady-state mRNA of the indicator gene by the compound correlated with reduction of the gene product in the constitutive cell line. Ro 24-7429 has broad activity against several strains of HIV-1 in different cell lines, peripheral blood lymphocytes, and macrophages (IC90 = 1-3 microM). Importantly, Ro 24-7429 inhibited viral replication in both acute and chronic infection in vitro, a characteristic expected of a Tat antagonist and not shared by viral reverse transcriptase inhibitors. Consistent with this, the compound reduced cell-associated viral RNA and proteins and partially restored cell-surface CD4 in chronically infected cells. After 2 years of continued weekly passage of the virus in fresh CEM cells grown in the presence of the compound at 1 or 10 microM, the virus did not develop resistance to the drug. These results indicate that the compound's action might involve a cellular factor.

Alkaline Phosphatase↗
PubMed1993

Uniform detection of HIV-1 in alveolar macrophages of pediatric but not adult AIDS patients.

Manifestations of pulmonary disease in pediatric AIDS patients differ from those in adults. To evaluate whether differences in the frequency of alveolar macrophage (AM) infection with human immunodeficiency virus type 1 (HIV-1) could account for these clinical distinctions, we undertook a comparative analysis of HIV-1 DNA in AMs from pediatric and adult AIDS patients by enzymatic amplification. A higher frequency of viral DNA detection in pediatric cases (100%) compared with adults (67%) was observed. The sensitivity of detection was 1 viral DNA copy per 4000 AMs; matched peripheral blood mononuclear cells from six of seven pediatric and eight of nine adult patients tested HIV-1 DNA positive. Adult but not pediatric patients exhibited a marked alveolar lymphocytosis, 32% mean lymphocyte count compared with 7.0%, respectively. These results suggest that the burden of HIV-1 in the lungs of pediatric AIDS patients is greater than that in adults.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Human immunodeficiency virus type 1 infection requires reverse transcription of nascent viral RNA.

We have previously shown that during human immunodeficiency virus type 1 (HIV-1) infection in vitro continued reverse transcription is required for stable HIV-1 production, but entry by progeny virus is not. To determine the source of the viral RNA reverse-transcribed late in infection, we employed inhibitors of HIV-1 transmission, reverse transcription, and proteolysis of the Gag-Pol polyprotein to interrupt HIV-1 infection in vitro. The kinetics of synthesis of viral DNA, RNA, and proteins was examined. During single-cycle infection, inhibition of reverse transcription 24-72 hr after infection delayed production of viral RNA and protein 10 days. Although viral DNA was detected in Southern blots, inhibition of Gag-Pol processing or transient inhibition of reverse transcription blocked its expression. We propose that after initial reverse transcription of input virion RNA is complete, newly synthesized HIV-1 RNA is reverse-transcribed before its export in virions to yield the viral DNA required for stable HIV-1 production.

Cells, Cultured↗
PubMed1993

Infection of macrophages with lymphotropic human immunodeficiency virus type 1 can be arrested after viral DNA synthesis.

Lymphotropic strains of human immunodeficiency virus type 1 (HIV-1), including HTLV-IIIB, replicate poorly in macrophages. We have shown previously that lymphotropic HIV-1 fuses equally well with T lymphocytes and macrophages (M. J. Potash, M. Zeira, Z.-B. Huang, T. Pearce, E. Eden, H. Gendelman, and D. J. Volsky, Virology 188:864-868, 1992), suggesting that events in the virus life cycle following virus-cell fusion limit virus replication. We report here that HIV-1 DNA is synthesized efficiently in either ADA or HTLV-IIIB infected alveolar macrophages or monocyte-derived macrophages within 24 h of virus infection, as observed by polymerase chain reaction for amplification of viral DNA sequences from the gag gene. Infection by a cloned lymphotropic HIV-1 strain, N1T-A, also leads to viral DNA synthesis. However, circular viral DNA was detected during strain ADA infection but not during HTLV-IIIB or N1T-A infection of monocyte-derived macrophages. These findings indicate that during replication of lymphotropic HIV-1 in macrophages, all steps of the virus life cycle up to and including reverse transcription take place and that defects in later events, including DNA migration to the nucleus, may account for the limited production of viral proteins.

DNA, Viral↗
PubMed1993

Human immunodeficiency virus type 1 DNA synthesis, integration, and efficient viral replication in growth-arrested T cells.

Human immunodeficiency virus type 1 (HIV-1) replicates efficiently in nonproliferating monocytes and macrophages but not in resting primary T lymphocytes. To determine the contribution of cell division to the HIV-1 replicative cycle in T cells, we evaluated HIV-1 expression, integration of proviral DNA, and production of infectious progeny virus in C8166 T-lymphoid cells blocked in cell division by treatment with either mitomycin, a DNA cross-linker, or aphidicolin, a DNA polymerase alpha inhibitor. The arrest of cell division was confirmed by assay of [3H]thymidine uptake; the nondividing cells remained viable for at least 3 days after treatment. HIV-1 was expressed and replicated equally well in nondividing and dividing C8166 cells, as judged by the comparison of the levels of p24 core antigens in culture supernatants, the proportion of cells expressing HIV-1 specific antigens, the pattern and quantity of HIV-1 DNA present in the extrachromosomal and total cellular DNA fractions, and the biological activity of progeny viruses. A polymerase chain reaction-based viral DNA integration assay indicated that HIV-1 provirus was integrated in C8166 cells treated with either of the two inhibitors of cell division. Similar results were obtained by using growth-arrested Jurkat T-lymphoid cells. We conclude that cell division and cellular DNA synthesis are not required for efficient HIV-1 expression in T cells.

Aphidicolin↗
PubMed1992

A Tat antagonist inhibits HIV-1 induction in naturally infected and experimentally infected T cells.

Ro 5-3335, a novel antagonist of human immunodeficiency virus type 1 (HIV-1) Tat activity, inhibits acute and chronic HIV-1 infection in T lymphocytes. Here we describe the effects of Ro 5-3335 on the accumulation of viral DNA during primary infection, the induction of virus from a latently infected cell line, and the expression of virus upon activation of naturally infected T cells. Ro 5-3335 permitted initial DNA synthesis during primary infection, but inhibited the subsequent increase in viral DNA copy number. The induction of HIV-1, as determined by the synthesis of p24 core antigen, was inhibited by 99% by Ro 5-3335 in both the model cell line and naturally infected T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Virus-cell membrane fusion does not predict efficient infection of alveolar macrophages by human immunodeficiency virus type 1 (HIV-1).

Alveolar macrophages (AM) are the principal target cells for HIV-1 in lung tissue. To investigate the mechanisms of HIV-1 infection and efficient replication in these cells we isolated AM from 14 HIV-1 negative donors and exposed them to two virus isolates, either N1T, which replicates well in T lymphocytic and monocytic cell lines, or ADA, a monocytotropic virus. Membrane fluorescence dequenching assays demonstrated that HIV-1/N1T fuses efficiently with AM plasma membranes at neutral pH and that this interaction requires cellular CD4. Despite efficient fusion, AM from eight of 14 donors were not susceptible to productive infection with N1T. In contrast, ADA replicated in all AM populations tested. Soluble CD4 blocked infection of AM by either N1T or ADA, indicating that, like membrane fusion, entry of infectious virus requires an interaction with cellular CD4. Analysis of HIV-1 DNA accumulation in infected cells by enzymatic amplification revealed that productive infection by ADA correlated with a high HIV-1 DNA copy number and abortive infection by N1T was characterized by little or no stable cDNA. These studies suggest that the differences between the two HIV-1 strains studied in their ability to replicate in AM reside in phases of the virus life cycle that follow virus-cell fusion.

CD4 Antigens↗
PubMed1992

Evaluation of multiple parameters of HIV-1 replication cycle in testing of AIDS drugs in vitro.

Evaluation of the activities of antiretroviral agents and an immunoregulatory compound has been made using two models of HIV-1 infection and three measurements of virus expression. Acute infection of Jurkat cells or chronic/inducible infection in U1.1 cells was monitored at multiple time points after drug treatment. The 50% effective concentrations (EC50) of the HIV-1 inhibitors suramin, 3'-azido-3'-deoxythymidine (AZT), and 2',3'-dideoxycytidine, as measured by HIV-1 RNA hybridization in Jurkat cells two days after infection, were comparable to EC50 values obtained in parallel measurements of extracellular p24 levels and percent HIV-1 IF-positive cells. However, these measurements diverged: at seven days after infection the EC50 of AZT was greater than 10 microM when intracellular HIV-1 RNA was assayed, 0.2 microM by IF, and 0.03 microM by p24 assay. Human thymic humoral factor displayed no direct inductive activity in chronic HIV-1 infection in U1.1 cells, while phorbol ester and lymphocyte supernatants induced all parameters. These observations warrant care when interpreting results of only a single assay and suggest that definitive assay of HIV-1 infection requires measurements of multiple parameters of virus expression.

Antiviral Agents↗
PubMed1992

Discovery and characterization of an HIV-1 Tat antagonist.

Ro 5-3335, 7-chloro-5-(2-pyrryl)-3H-1,4-benzo-diazepin-2-(H)-one, has been shown to inhibit gene expression controlled by the human immunodeficiency virus-1 (HIV-1) LTR promoter. The inhibition was specific for the viral transcriptional transactivator Tat. The compound did not inhibit the basal activity of the HIV-1 LTR or the activity of promoters not responsive to Tat. Consistent with its mode of action, Ro 5-3335 inhibited HIV-1 replication (IC50 = 0.1-1 microM) by reducing viral RNA synthesis in acutely, as well as chronically, infected cells in vitro. The compound was active against HIV-1 and HIV-2, and AZT-resistant clinical isolates.

Animals↗
PubMed1991

Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist.

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

Antiviral Agents↗