Search PubMed⌕ Search

Biomedical subjects

R J Pomerantz

Publications and source records attributed to R J Pomerantz.

At least 91 records · Page 5Linked to original sources
PubMed1996

Kinetic analysis of intravirion reverse transcription in the blood plasma of human immunodeficiency virus type 1-infected individuals: direct assessment of resistance to reverse transcriptase inhibitors in vivo.

Intravirion reverse transcripts have been identified in the blood plasma of human immunodeficiency virus type 1 (HIV-1)-infected individuals. In the present studies, the kinetic processes of intravirion HIV-1 reverse transcription, in the blood plasma of HIV-1-infected persons treated with nevirapine, were investigated. Nevirapine is a nonnucleoside inhibitor of reverse transcriptase (RT) which decreases the level of HIV-1 viral particles in the blood plasma of infected individuals. By analyzing HIV-1 virions at different time points prior to and after initiation of nevirapine therapy in vivo, the levels of intravirion reverse transcripts have been demonstrated to be dramatically susceptible to this anti-RT agent, out of proportion to effects on plasma virion load. The intravirion reverse transcripts were also documented to rebound to the pretreatment levels, concomitant with the development of resistant viral mutants. In addition, the infectivity of HIV-1 virions dramatically decreased after nevirapine treatment, further indicating that the effects of this anti-RT agent begin within the cell-free virions. Since the levels of intravirion reverse transcripts were altered according to the susceptibility or resistance of the HIV-1 RT enzyme to this inhibitor, these data demonstrate that the formation of intravirion reverse transcripts is a dynamic process in vivo. Moreover, because the alteration in ratios between intravirion HIV-1 reverse transcripts and viral genomic RNA directly reflects the efficiency of reverse transcription, we propose that the determination of these ratios in the blood plasma of HIV-1-positive patients may be a useful and, most importantly, a direct assay to monitor the efficacy of anti-RT agents in vivo.

Base Sequence↗
PubMed1996

Intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle by targeting human immunodeficiency virus type 1 integrase.

Integration of viral DNA into a chromosome of the infected host cell is required for efficient replication of a retroviral genome, and this reaction is mediated by the virus-encoded enzyme integrase (IN). As IN plays a pivotal role in establishing infection during the early stages of the retroviral life cycle, it is an attractive target for therapeutic intervention. However, the lack of effective antiviral drug therapy against this enzyme has led to the testing of other novel approaches towards its inhibition. In these studies, a panel of anti-human immunodeficiency virus type 1 (anti-HIV-1) IN hybridomas has been used in the construction of single-chain variable antibody fragments (SFvs). The monoclonal antibodies produced by these hybridomas, and derived SFvs, bind to different domains within IN. We now demonstrate that intracellular expression of SFvs which bind to IN catalytic and carboxy-terminal domains results in resistance to productive HIV-1 infection. This inhibition of HIV-1 replication is observed with SFvs localized in either the cytoplasmic or nuclear compartment of the cell. The expression of anti-IN SFvs in human T-lymphocytic cells and peripheral blood mononuclear cells appears to specifically neutralize IN activity prior to integration and, thus, has an effect on the integration process itself. These data support our previous studies with an anti-HIV-1 reverse transcriptase SFv and demonstrate further that intracellularly expressed SFvs can gain access to viral proteins of the HIV-1 preintegration complex. This panel of anti-HIV-1 IN SFvs also provides the tools with which to dissect the molecular mechanism(s) directly involved in integration within HIV-1-infected cells.

Amino Acid Sequence↗
PubMed1996

Nuclear preservation and cytoplasmic degradation of human immunodeficiency virus type 1 Rev protein.

Rev, a major regulatory protein of human immunodeficiency virus type 1, has been demonstrated to shuttle between the nucleus and cytoplasm of infected cells. The fate of the Rev protein in living cells was evaluated by pulse-chase experiments using a transient Rev expression system. Sixteen hours after chasing with unlabelled amino acids, 45% of the labelled Rev was still present, which clearly indicates a long half-life of Rev in living cells. A Rev mutant which is deficient in the ability to migrate from the nucleus to the cytoplasm was degraded more slowly than the wild-type Rev protein. As well, another Rev mutant protein, which lacks a functional nucleolar targeting signal (NOS) and is unable to enter the cell nucleus, was rapidly degraded and undetectable 16 h after chasing. Nuclear-nucleolar targeting properties provided by a divergent NOS from a related retrovirus, which was used to substitute for the NOS of Rev, increased the intracellular half-life of this Rev mutant. Moreover, coexpression of an intracellular anti-Rev single-chain antibody (SFv), which has been shown to interfere with the nuclear translocation of Rev, accelerated the degradation of the wild-type Rev protein. Differential degradation of Rev in the nucleus and cytoplasm may play a critical role in determining and maintaining different stages of human immunodeficiency virus type 1 infection, in conjunction with the shuttling properties of the Rev protein.

Amino Acid Sequence↗
PubMed1996

Endogenous reverse transcription of human immunodeficiency virus type 1 in physiological microenviroments: an important stage for viral infection of nondividing cells.

Endogenous reverse transcription (ERT) of retroviruses has long been considered a somewhat artificial process which only mimics reverse transcription occurring in target cells, as detergents or amphipathic peptides have classically been used to make the envelopes of retroviruses in these reaction systems permeable. Recently, several studies suggested that ERT of human immunodeficiency virus type 1 (HIV-1) might occur without detergent treatment. However, this phenomenon could be due to damage of the retroviral envelope during the process of virion purification or freezing and thawing. In this report, intravirion HIV-1 ERT, without detergent-induced permeabilization, is demonstrated to occur in the natural microenvironments of HIV-1 virions and is not caused by artificial processes. Therefore, this stage of the viral life cycle was termed natural ERT (NERT). The efficiency of NERT in HIV-1 virions was markedly augmented by several physiological substances in the extracellular milieu, such as polyamines and deoxyribonucleoside triphosphates. In addition, HIV-1 virions in seminal plasma samples harbored dramatically higher levels of full-length or nearly full-length reverse transcripts than virions isolated from peripheral blood plasma samples of HIV-1-seropositive men. When HIV-1 virions were incubated with seminal plasma samples, infectivity in initially nondividing cells was also significantly enhanced. Thus, we suggest that HIV-1 virions are actively altered by the extracellular microenvironment and that NERT may play an important role in viral infection of nondividing cells.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Binding of intracellular anti-Rev single chain variable fragments to different epitopes of human immunodeficiency virus type 1 rev: variations in viral inhibition.

Intracellular immunization to target the human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev has been explored as a genetic therapy for AIDS. Efficient intracellular expression of rearranged immunoglobulin heavy and light chain variable regions of anti-Rev monoclonal antibodies, with various vectors, and subsequent inhibition of HIV-1 replication have been previously reported by our laboratories. To further understand the molecular mechanisms and effects that intracellular anti-Rev single chain variable fragments (SFvs) have against HIV-1, via blocking of Rev function, two anti-Rev SFvs which specifically bind to differing epitopes of the Rev protein have been cloned. One SFv binds to the Rev activation domain, and the second SFv binds to the distal C terminus of Rev in the nonactivation region. Further studies now demonstrate that both anti-Rev SFvs lead to variable resistance to HIV-1 infection. Although binding affinity assays demonstrated that the SFv which specifically recognizes the Rev activation domain (D8) had an extracellular binding affinity significantly lower than that of the SFv specific to the nonactivation region (D1O), the SFv D8 demonstrated more potent activity in inhibiting virus production in human T-cell lines and peripheral blood mononuclear cells than did SFv D10. Thus, extracellular binding affinities of an SFv for a target viral protein cannot be used to directly predict its activity as an intracellular immunization moiety. These data demonstrate potential approaches for intracellular immunization against HIV-1 infection, by efficiently blocking specific motifs of Rev to after the function of this retroviral regulatory protein. These studies extend the understanding of the effects, on a molecular level, of SFvs binding to critical epitopes of Rev and further suggest that rational design of SFvs, with interactions involving specific viral moieties which mediate HIV-1 expression, may hold promise for the clinical application of genetic therapies to combat AIDS.

Amino Acid Sequence↗
PubMed1996

Targeting human immunodeficiency virus type 1 reverse transcriptase by intracellular expression of single-chain variable fragments to inhibit early stages of the viral life cycle.

Novel molecular approaches to inhibit human immunodeficiency virus type 1 (HIV-1) infection have received increasing attention because of the lack of effective antiviral drug therapies in vivo. We now demonstrate that cells can be intracellularly immunized by cytoplasmic expression of single-chain variable antibody fragments (SFv) which bind to the HIV-1 reverse transcriptase (RT) enzyme. The expression of anti-RT SFv in T-lymphocytic cells specifically neutralizes the RT activity in the preintegration stage and affects the reverse transcription process, an early event of the HIV-1 life cycle. Blocking the virus at these early stages dramatically decreased HIV-1 propagation, as well as the HIV-1-induced cytopathic effects in susceptible human T lymphocytes, by impeding the formation of the proviral DNA. These data also demonstrate that intracellular, complete SFvs may gain access to viral proteins of the HIV-1 preintegration complex. These SFvs will provide a tool with which to better understand the molecular mechanisms involved in restricting viral replication in HIV-1-infected cells.

Animals↗
PubMed1996

Evaluation of relative promoter strengths of the HIV-1-LTR and a chimeric RSV-LTR in T lymphocytic cells and peripheral blood mononuclear cells: promoters for anti-HIV-1 gene therapies.

Gene therapy approaches for human immunodeficiency virus type 1 (HIV-1) infections focus on the transfer of critical genetic elements into CD4+ T lymphocytes and CD34+ stem cells. Ideally, expression of the anti-HIV-1 gene constructs should be induced during early stages of infection to combat high turnover of the replicating virus. In this study, we investigated the activity of two promoters, HIV-1 long terminal repeat (HIV-1-LTR) and Rous sarcoma virus (RSV) LTR fused with the transactivation response element (TAR) from the HIV-1-LTR (ie RSV-TAR) in presence of Tat, the major HIV-1 transcriptional transactivator and an early gene product in HIV-1 infection. Comparative expression from both of these plasmids was analyzed by measuring expression of a reporter gene, chloramphenicol acetyltransferase (CAT), after transfection of the promoter-CAT constructs and a Tat-expressing plasmid into CEM T lymphocytic cells and peripheral blood mononuclear cells (PBMC). The HIV-1-LTR could be transactivated by Tat in both unstimulated and stimulated cells. Although the RSV-TAR had a relatively high basal level of expression, Tat transactivation of this chimeric promoter occurred only in unstimulated cells. These results suggest that the HIV-1-LTR may be a better promoter for therapeutic gene expression in anti-HIV-1 intracellular immunization approaches.

Avian Sarcoma Viruses↗
PubMed1995

Sodium butyrate stimulation of HIV-1 gene expression: a novel mechanism of induction independent of NF-kappa B.

Nuclear factor-kappa B (NF-kappa B) has been shown to play a central role in stimulating human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR)-directed viral gene expression. We have previously described a cell line (TE671/RD) that fails to respond to phorbol myristate acetate (PMA) or tumor necrosis factor-alpha (TNF-alpha) in terms of amplifying HIV-1 LTR-driven gene expression unless it is concurrently treated with sodium butyrate. It was not determined whether this lack of response stemmed from an inability of these cells to produce free NF-kappa B or from ineffectual interaction of this sequence-specific transcriptional factor with its target. We now show that these cells are in fact capable of inducing a free nuclear NF-kappa B-binding activity when stimulated with PMA but not when treated with sodium butyrate alone. Furthermore, we show that sodium butyrate alone is equally potent in stimulating HIV-1 LTR-directed gene expression in latently infected U1 and ACH-2 cells in the absence of induction of nuclear NF-kappa B, as compared with PMA, which induces NF-kappa B activation in these cells. We also show that stimulation of HIV-1 expression in U1 cells with sodium butyrate is not blocked by N-acetylcysteine, whereas that of PMA stimulation is blocked. These observations are discussed in the context of a model where chromatin structure participates in the maintenance of restricted HIV-1 viral gene expression in these cells.

Acetylcysteine↗
PubMed1995

A CNS-enriched factor that binds to NF-kappa B and is required for interaction with HIV-1 tat.

The Human Immunodeficiency Virus type 1 (HIV-1) Tat protein is a potent activator of transcription directed by the long terminal repeat (LTR), an essential step in the life-cycle of HIV-1. While interaction of Tat with an RNA element encoded by downstream LTR sequences (termed TAR) is commonly considered essential to activation, numerous recent reports have implicated upstream transcription elements within the LTR as participants in mediating this activation. We have recently demonstrated that Tat activation occurs independent of the TAR element in certain cells derived from the central nervous system (CNS), and that this activation is mediated by the kappa B domain of the LTR. Further, CNS-derived cells were found to contain kappa B-binding activity capable of both interacting with Tat and activating LTR transcription in vitro. The present study demonstrates that the kappa B-binding transcription factor derived from CNS cells consists of a component indistinguishable from prototypical Nuclear Factor-kappa B (NF-kappa B) (in size, mobility on native gel, kinetics of activation and cognate binding sequence) as well as a supershifting component that is dissociable under certain conditions. The supershifting activity is found to stabilize binding of the presumed NF-kappa B to DNA. Further, only the form of NF-kappa B which is associated with this supershifting activity is capable of binding Tat. We hypothesize a model in which Tat utilizes this interaction to activate HIV-1 through the NF-kappa B domain of the LTR in circumstances where TAR is absent. This model has implications with respect to the ability of Tat to alter cellular gene expression and perhaps contribute to the array of problems seen in HIV-1 infection such as altered immune status, CNS toxicity, and the formation of tumors.

Astrocytes↗
PubMed1995

Intracellular immunization against HIV-1 infection of human T lymphocytes: utility of anti-rev single-chain variable fragments.

Genetic therapy offers a potentially promising approach with which to combat human immunodeficiency virus type 1 (HIV-1) infections. Several modalities, using protein- and RNA-based systems, have recently been shown to inhibit HIV-1 replication. A single-chain variable fragment (SFv), constructed from the cDNA of a monoclonal antibody to the HIV-1 regulatory protein Rev, has been demonstrated to potently inhibit HIV-1 replication, when expressed intracellularly in an epithelial cell-line (HeLa-CD4). Murine retroviral shuttle vectors, which express the anti-Rev SFv moiety, have now been constructed. HIV-1 infection was dramatically inhibited in human T-lymphocytic cell-lines, CEM and Sup-T1, transduced with these anti-Rev SFv-expressing vectors. This resistance to high levels of HIV-1 expression was demonstrated in both mixed populations and clones of these cells. Of further potential clinical significance, HIV-1 infection was also potently inhibited in human peripheral blood mononuclear cells (PBMC), transduced with retroviral vectors expressing the anti-Rev SFv molecule. These data suggest that intracellular expression of anti-Rev SFvs, or related approaches, may be utilized as genetic therapy, or intracellular immunization, for HIV-1 infections in vivo.

Animals↗
PubMed1995

Infection of oral mucosal cells by human immunodeficiency virus type 1 in seropositive persons.

Molecular studies have revealed significant amounts of human immunodeficiency virus type 1 (HIV-1) provirus DNA in saliva of HIV-infected persons. However, cellular localization has not been determined. In situ polymerase chain reaction (IS-PCR) was done on saliva-associated cells for localization of HIV-1 provirus DNA. Results indicate its presence in the nuclei of saliva-associated epithelial cells in 29 (83%) of 35 HIV-1-seropositive subjects. In 24 (83%) of the 29 IS-PCR-positive samples, 0.1%-4.0% of the mucosal epithelial cells exhibited nuclear localization of HIV-1 DNA. In addition, HIV-1 provirus DNA was detected in monocytes or lymphocytes of all salivary samples from the 35 subjects. The localization of HIV-1 provirus DNA indicates that epithelial cells are another cell type infected by HIV-1 in vivo. These findings suggest epithelial cells in other body sites might also be infected with HIV-1.

Adult↗
PubMed1995

Evidence that a cell cycle regulator, E2F1, down-regulates transcriptional activity of the human immunodeficiency virus type 1 promoter.

Proliferation of eukaryotic cells is orchestrated by a series of cellular proteins which participate in various stages of the cell cycle to guide the cell through mitosis. Some of these proteins, including E2F1, play a critical role in G1 and S phases by coordinately regulating expression of several important cell cycle-associated genes. On the basis of recent observations indicating a block in human immunodeficiency virus type 1 (HIV-1) replication in cells arrested in G1/S phase of the cell cycle, we sought to evaluate the regulatory action of E2F1 on transcription from the HIV-1 long terminal repeat (LTR). Results from transient transfection of cells with an E2F1 expression plasmid indicated that E2F1 has the ability to suppress basal transcriptional activity of the LTR and to diminish the extent of the Tat-induced activation of the viral promoter. Deletion analysis of the HIV-1 LTR in transfection studies revealed the presence of two major elements responsive to E2F1 repression located distally (-454 to -381) and proximally (-117 to -80) with respect to the +1 transcription start site. E2F1-mediated suppression of LTR activity was observed in a wide range of human cell lines. Expression of E2F1 by a transgene showed an inhibitory effect on the levels of reverse transcriptase activity obtained upon introduction of the proviral genome into cells. The data presented in this study suggest that cellular regulatory proteins involved in the progression of cells through the mitotic cycle could play crucial roles in determining the efficiency of HIV-1 replication during the various stages of infection. The possible roles of these factors in viral latency and activation are discussed.

Carrier Proteins↗
PubMed1995

Increasing transduction efficiency of recombinant murine retrovirus vectors by initiation of endogenous reverse transcription: potential utility for genetic therapies.

Reverse transcription of retroviral genomic RNA in a target cell is influenced by cellular factors, including the concentration of deoxyribonucleoside triphosphates (dNTPs). In addition, recent data have demonstrated that reverse transcription can be driven within human immunodeficiency virus type 1 virions, prior to infection of a cell, by increasing extracellular concentrations of dNTPs. In attempts to increase the transduction efficiency of recombinant murine leukemia virus vectors, endogenous reverse transcription was initiated within cell-free, recombinant murine leukemia virus virions in the presence of relatively high concentrations of dNTPs. As a result, the expression of transduced genes via these retroviral vectors was increased approximately 10-fold by treatment of virions with dNTPs. Combined with our previous data, these observations suggest that virion-associated DNA synthesis can occur in diverse groups of retroviruses and positively alter retroviral infectivity. As such, these manipulations may be useful for increasing the efficiency of retrovirus-mediated gene delivery.

Animals↗
PubMed1994

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA.

Animals↗
PubMed1994

Potent inhibition of human immunodeficiency virus type 1 replication by an intracellular anti-Rev single-chain antibody.

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

Amino Acid Sequence↗
PubMed1994

Tat and rev differentially affect restricted replication of human immunodeficiency virus type 1 in various cells.

Human immunodeficiency virus type 1 (HIV-1) can infect various cell lines in culture and be maintained in a chronic state of restricted replication. These states of proviral latency are characterized by a predominance of spliced compared to unspliced viral RNA species. The proximate molecular mechanisms leading to restricted HIV-1 replication may differ in various cell lines. Importantly, recent studies have demonstrated that the site of integration is the critical parameter leading to proviral latency in ACH-2 cells. Utilizing murine retroviral shuttle vectors, the HIV-1 Tat protein was demonstrated to dramatically increase HIV-1 expression in the restrictively infected U1 monocytic cell line but not in the ACH-2 T-lymphocytic line. The HIV-1 Rev protein only modestly increased viral expression in both of these cell types. Thus, these data support the hypothesis that the mechanisms which initiate and/or maintain restricted HIV-1 expression may differ in various cell types in cell culture, and possibly in vivo.

Base Sequence↗