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Biomedical subjects

R J Pomerantz

Publications and source records attributed to R J Pomerantz.

At least 55 records · Page 3Linked to original sources
PubMed1999

A genetically engineered spleen necrosis virus-derived retroviral vector that displays the HIV type 1 glycoprotein 120 envelope peptide.

We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.

Animals↗
PubMed1999

Human immunodeficiency virus type 1 Vpr alters bone marrow cell function.

Vpr, a 96 amino acid protein, encoded by the human immunodeficiency virus type I (HIV-1), is important for efficient infection of mononuclear phagocytic cells. These cells are abundant in whole bone marrow, which can easily be cultured in vitro to support hematopoiesis. Our experiments indicate that Vpr plays a role in the potent activation of murine and human mononuclear phagocytic cells within a hematopoietic microenvironment. In murine cultures, avid erythrophagocytosis is triggered by transduction of marrow cells with supernatant derived from PA317 cells transfected with a murine retroviral delivery vector bearing a Vpr expression cassette. Supernatants derived from cells transfected with the same vector carrying sequences for the expression of other relevant viral and nonviral proteins do not induce erythrophagocytosis to any marked degree. The effect on human marrow cells is similar, where treatment promotes adhesion of mononuclear phagocytic cells to culture plates in association with other nucleated and nonnucleated cells that undergo subsequent engulfment. The differential effects of Vpr point and deletion mutants in both marrow culture systems fortify the view that the effect is specific to HIV-1 Vpr. Addition of low molar quantities of purified Vpr to marrow cultures is also capable of promoting cell adhesion and phagocytosis, suggesting that extracellular Vpr is the effector of the phenomenon. Accelerated phagocytosis is a hallmark of promonocyte, monocyte, and macrophage activation and its occurrence within a hematopoietic microenvironment may account for critical in vivo pathogenic features of HIV-1 infection. First, activation of mononuclear phagocytes may promote productive viral infection; and second, premature phagocytosis could provide, at least in part, a molecular explanation for the induction of the idiopathic cytopenias that are typical of individuals infected with HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1999

A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions.

A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN.

Artificial Gene Fusion↗
PubMed1999

Nuclear and nucleolar targeting of human ribosomal protein S25: common features shared with HIV-1 regulatory proteins.

The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-1 cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This configuration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunodeficiency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the first NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three different backbones with RPS25 cDNA fragments. Immunofluorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/nucleolar targeting signal. The identified peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNA-binding transactivators.

Animals↗
PubMed1999

HIV-1 virions produced from replicating peripheral blood lymphocytes are more infectious than those from nonproliferating macrophages due to higher levels of intravirion reverse transcripts: implications for pathogenesis and transmission.

It has been demonstrated that intravirion reverse transcription in human immunodeficiency virus type I (HIV-1) occurs in the presence of physiological substances and that the intravirion HIV-1 reverse transcripts are important for the establishment of infection in nondividing cells. In this report, we demonstrate that the infectivity of the virus produced from replicating peripheral blood lymphocytes (PBL) is significantly higher than that of virions produced by nonproliferating macrophages, upon infection of either initially quiescent PBLs or macrophages. This directly correlated with significantly higher intravirion reverse transcripts in the virions from replicating PBLs, compared to those from macrophages. Treatment of replicating PBLs and macrophages with 3'- azido-3'- deoxythymidine resulted in decreased intravirion reverse transcripts and lower infectivity of produced virions, upon infection of initially quiescent T-cells or macrophages. Because both nonproliferating macrophages and replicating lymphocytes are the major reservoirs for HIV-1 in vivo, we propose that as the result of higher levels of intravirion reverse transcripts, the HIV-1 virions produced from actively replicating cells are more infectious than those from nonproliferating cells. As such, this scenario implies at least one molecular mechanism for the intra- and interhost transmission of HIV-1.

CD4-Positive T-Lymphocytes↗
PubMed1999

Ribozyme-mediated specific gene replacement of the alpha1-antitrypsin gene in human hepatoma cells.

BACKGROUND/AIMS: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be "gene replacement" accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in alpha1-antrypsin (alpha1-AT) deficiency is caused by the lack of alpha1-AT function, the liver involvement is due to the accumulation of the mutated alpha1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of alpha1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha1-AT protein in the liver. METHODS: For the inhibition of alpha1-AT gene expression, panels of alpha1-AT-specific hammerhead ribozymes designed to target different GUC sites in the alpha1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional alpha1-AT ribozyme and was combined with a modified alpha1-AT gene, whose product was engineered to be resistant to the specific alpha1-AT ribozyme. This construct was transduced into target hepatoma cells. RESULTS: The transduced hepatoma cells showed the effective expression of modified alpha1-AT, under the conditions where the endogenous alpha1-AT gene expression was inhibited. CONCLUSION: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of alpha1-AT deficiency.

Base Sequence↗
PubMed1999

Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection.

Human immunodeficiency virus type 1 (HIV-1) encodes several proteins that are packaged into virus particles. Integrase (IN) is an essential retroviral enzyme, which has been a target for developing agents to inhibit virus replication. In previous studies, we showed that intracellular expression of single-chain variable antibody fragments (SFvs) that bind IN, delivered via retroviral expression vectors, provided resistance to productive HIV-1 infection in T-lymphocytic cells. In the current studies, we evaluated simian-virus 40 (SV40) as a delivery vehicle for anti-IN therapy of HIV-1 infection. Prior work suggested that delivery using SV40 might provide a high enough level of transduction that selection of transduced cells might be unnecessary. In these studies, an SV40 expression vector was developed to deliver SFv-IN (SV(Aw)). Expression of the SFv-IN was confirmed by Western blotting and immunofluorescence staining, which showed that > 90% of SupT1 T-lymphocytic cells treated with SV(Aw) expressed the SFv-IN protein without selection. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression and syncytium formation, was potently inhibited in cells expressing SV40-delivered SFv-IN. Levels of inhibition of HIV-1 infection achieved using this approach were comparable to those achieved using murine leukemia virus (MLV) as a transduction vector, the major difference being that transduction using SV40 did not require selection in culture whereas transduction with MLV did require selection. Therefore, the SV40 vector as gene delivery system represents a novel therapeutic strategy for gene therapy to target HIV-1 proteins and interfere with HIV-1 replication.

Blotting, Western↗
PubMed1999

Role of lentiviral lytic polypeptide I (LLP-I) in the selective cytotoxicity of gamma-glutamylcysteine ethyl ester against human immunodeficiency virus type 1-producing cells.

Selective cytotoxic effects of gamma-glutamylcysteine ethyl ester (gamma-GCE) against human immunodeficiency virus type 1 (HIV-1)-infected H-9 T lymphocytic cells were demonstrated previously. However, the mechanism of those effects remained unclear. Here, we report on enhanced cytotoxicity of the lentiviral lytic peptide I (LLP-I) of gp41, the envelope transmembrane glycoprotein of HIV-1, in the presence of gamma-GCE. Without gamma-GCE, the cytotoxic effect of LLP-I was transient, whereas with gamma-GCE, cell death induced by LLP-I remained continuous until termination. Of note, such effects by gamma-GCE were also observed with another unrelated amphipathic peptide toxin, melittin. These results suggest that the synergistic cytotoxic effect of gamma-GCE and LLP-I may play a central role in the molecular mechanism of the selective cytotoxicity of gamma-GCE in HIV-1-infected T lymphocytic cells.

Anti-HIV Agents↗
PubMed1998

Human immunodeficiency virus type 1 in the semen of men receiving highly active antiretroviral therapy.

BACKGROUND: Highly active antiretroviral therapy can effectively decrease the levels of human immunodeficiency virus type 1 (HIV-1) virions in peripheral plasma and seminal fluid of infected men. Whether the genital tract of HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable virus in the peripheral plasma harbors replication-competent virus is not known. METHODS: We collected peripheral-blood and semen samples from seven men with HIV-1 infections who were receiving highly active antiretroviral therapy and who had no detectable viral RNA (fewer than 50 copies per milliliter) in plasma and analyzed the samples for cell-associated proviral DNA using a quantitative polymerase-chain-reaction assay. Replication-competent viruses were evaluated by cell-coculture assays. Proviral DNA and replication-competent virus obtained from peripheral-blood and seminal cells were also analyzed by sequencing relevant viral genes. RESULTS: Despite the long-term suppression of HIV-1 RNA in the plasma of the seven men, proviral DNA was detected in seminal cells in four. Replication-competent viruses were recovered from peripheral-blood cells in three men and from the seminal cells in two of these three men. The viruses recovered from the seminal cells had no genotypic mutations suggestive of resistance to antiretroviral drugs and were macrophage-tropic, a feature that is characteristic of HIV-1 strains that are capable of being sexually transmitted. CONCLUSIONS: In HIV-1-infected men who are receiving highly active antiretroviral therapy and who have no detectable levels of viral RNA in plasma the virus may be present in seminal cells and therefore may be capable of being transmitted sexually.

Amino Acid Sequence↗
PubMed1998

Inhibition of HIV type 1 replication in chronically infected monocytes and lymphocytes by retrovirus-mediated gene transfer of anti-Rev single-chain variable fragments.

We investigated a strategy for gene therapy, intracellular expression of anti-HIV-1 Rev single-chain variable fragments (SFvs), in promonocytic (U1) and T (ACH-2) cell lines latently infected with HIV-1. The cellular and molecular mechanisms leading to activation of latent integrated HIV-1 provirus in U1 and ACH-2 cells have been well delineated. These cells produce HIV-1 in response to stimulation with certain cytokines. U1 and ACH-2 cells were transduced with a murine retroviral shuttle vector that expresses anti-Rev SFv (pLXSN-D8SFv-Rev) or with a control murine leukemia virus (MLV) vector (pLXSN). Tumor necrosis factor alpha (TFNalpha)-, interleukin 6 (IL-6)-, and phorbol myristate acid (PMA)-induced HIV-1 expression, as determined by reverse transcriptase (RT) assay, was significantly inhibited in cells transduced with pLXSN-D8SFv-Rev, compared with cells transduced with pLXSN. In addition, pLXSN-D8SFv-Rev-transduced cells, when incubated with monokine-enriched supernatants of human peripheral blood monocyte cultures, produced significantly less HIV-1 than did cells transduced with pLXSN. This resistance to cytokine-induced HIV-1 expression was demonstrated in SFv-transduced U1 and ACH-2 cells maintained in G418-free medium for 2 months. These data suggest that feasibility of utilizing various anti-HIV-1 SFvs to block activation of HIV-1 infection in vivo.

Anti-HIV Agents↗
PubMed1998

How HIV resists eradication.

Successfully treated patients with no evidence of plasma viremia may continue to harbor stable reservoirs of HIV at multiple sites, including those shielded by blood-tissue barriers. HIV may be controllable, but few believe that it can be eradicated with the limited group of agents now available. Creative new pharmacologic strategies are needed, as well as increased commitment to the development of a safe and effective vaccine.

Anti-HIV Agents↗
PubMed1998

A cis-acting peptide signal in human immunodeficiency virus type I Rev which inhibits nuclear entry of small proteins.

A peptide signal, which may control nucleo-cytoplasmic protein trafficking, was newly identified in human immunodeficiency virus type I (HIV-1) Rev, a lentiviral post-transcriptional transactivator. The sequence, in the amino-terminal portion of HIV-1 Rev, maintains a Rev mutant with a dysfunctional nuclear/nucleolar targeting signal outside of the nucleus, although this Rev molecule itself is small enough to pass through the nuclear pores. Transition of this sequence to the N-terminus of human T-lymphocytic leukemia/lymphoma virus type I (HTLV-I) p21x, which is usually located evenly distributed throughout the cell, resulted in capture of p21x in the cytoplasm. Mutational analysis clarified that a 14 residue peptide sequence was sufficient to display this inhibitory effect against nuclear entry. Furthermore, this HIV-1 Rev sequence was capable of inhibiting nuclear entry of a fragment of a human ribosomal protein, when it was fused to the carboxy terminus. The identified nuclear entry inhibitory signal (NIS) contains a conserved hydrophilicity motif, which forms an amphipathic helix. Significantly, this motif and its helical structure were shown to be important for NIS function and the HIV-1 Rev function itself. Possible roles for NIS as a molecular anchor are proposed herein.

Amino Acid Sequence↗
PubMed1998

Diversity of HIV-1 Vpr interactions involves usage of the WXXF motif of host cell proteins.

Targeting protein or RNA moieties to specific cellular compartments may enhance their desired functions and specificities. Human immunodeficiency virus type I (HIV-1) encodes proteins in addition to Gag, Pol, and Env that are packaged into virus particles. One such retroviral-incorporated protein is Vpr, which is present in all primate lentiviruses. Vpr has been implicated in different roles within the HIV-1 life cycle. In testing a new hypothesis in which viral proteins are utilized as docking sites to incorporate protein moieties into virions, we used the peptide phage display approach to search for Vpr-specific binding peptides. In the present studies, we demonstrate that most of the peptides that bind to Vpr have a common motif, WXXF. More importantly, we demonstrate that the WXXF motif of uracil DNA glycosylase is implicated in the interaction of uracil DNA glycosylase with Vpr intracellularly. Finally, a dimer of the WXXF motif was fused to the chloramphenicol acetyl transferase (CAT) gene, and it was demonstrated that the WXXF dimer-CAT fusion protein construct produces CAT activity within virions in the presence of Vpr as a docking protein. This study provides a novel potential strategy in the targeting of anti-viral agents to interfere with HIV-1 replication.

Amino Acid Sequence↗
PubMed1998

Modulation of HTLV-I gene expression by HIV-1 Rev through an alternative RxRE-independent pathway mediated by the RU5 portion of the 5'-LTR.

The 5'-RU5 portion of human T-lymphocyte virus type I (HTLV-I) long terminal repeat (LTR) had been reported to contain cis-acting elements for the controlled viral gene expression by the rex gene product. In this study, the human immunodeficiency virus type I (HIV-1) Rev protein was found to enhance gene expression, acting through the 5'-RU5 portion of HTLV-I, while the Rex-responsive element (RxRE)-mediated activation by Rev was reconfirmed to be negative. This positive action of HIV-1 Rev on HTLV-I gene expression seemed to be distinct from the widely accepted Rex or Rev function to facilitate the nuclear export of RxRE-containing unspliced viral mRNAs, since a trans-dominant, nuclear export-deficient mutant (RevM10) still retained the RU5-mediated effector function. Analyses of the functional aspects of Tat/Rev fusion proteins on the HTLV-I RU5 suggested a specific interaction of Rev and RU5, but lacked evidence for the binding of Rev to the RU5 at the RNA level. These results suggest an answer to the controversy regarding a Rex-like function occasionally observed with HIV-1 Rev and its related proteins. It may also be suggested that particular care should be taken when such a trans-dominant Rev mutant is considered to be used as a genetic therapy against HIV-I infection, in individuals infected with both HIV-I and HTLV-1.

Animals↗
PubMed1998

Natural endogenous reverse transcription of HIV-1.

Mechanisms involved with human immunodeficiency virus type I (HIV-1) sexual transmission are not fully defined. We have demonstrated that endogenous reverse transcription of lenti-viruses can occur within the intact virion. This takes place before direct infection of the target cells. In a biochemically active process, endogenous reverse transcription occurs in HIV-1 virions in specific microenvironments. In virions without non-physiological permeabilization, endogenous reverse transcription can occur and has been entitled natural endogenous reverse transcription (NERT). This molecular mechanism dramatically increases HIV-1 infection in initially-quiescent peripheral blood mononuclear cells, as well as non-proliferating cells such as macrophages. This molecular process may augment sexually transmission of HIV-1, as HIV virion particles in genital secretions are shown to have increased endogenous reverse transcripts and NERT is potently stimulated. Further studies are necessary to determine whether this molecular mechanism is critical in vivo for sexual transmission of this human lenti-viral agent.

Animals↗