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Biomedical subjects

Roger J Pomerantz

Publications and source records attributed to Roger J Pomerantz.

At least 55 records · Page 3Linked to original sources
PubMed2004

Antiviral effects of human immunodeficiency virus type 1-specific small interfering RNAs against targets conserved in select neurotropic viral strains.

RNA interference, a natural biological phenomenon mediated by small interfering RNAs (siRNAs), has been demonstrated in recent studies to be an effective strategy against human immunodeficiency virus type 1 (HIV-1). In the present study, we used 21-bp chemically synthesized siRNA duplexes whose sequences were derived from the gp41 gene, nef, tat, and rev regions of viral RNA. These sequences are conserved in select neurotropic strains of HIV-1 (JR-FL, JR-CSF, and YU-2). The designed siRNAs exerted a potent antiviral effect on these HIV-1 strains. The antiviral effect was mediated at the RNA level (as observed by the down-regulation of the HIV-1-specific spliced transcript generating a 1.2-kbp reverse transcription [RT]-PCR product) as well as viral assembly on the cell membrane. Spliced transcripts (apart from the most abundant transcript generating a 1.2-kbp RT-PCR product) arising from an unspliced precursor likely contributed, albeit to a lesser extent, to the antiviral effect. The resultant progeny viruses had infectivities similar to that of input virus. We therefore conclude that these siRNAs interfere with the processing of the unspliced transcripts for the gp41 gene, tat, rev, and nef, eventually affecting viral assembly and leading to the overall inhibition of viral production. Apart from using the gp41 gene as a target, the conservation of each of these targets in the above-mentioned viral strains, as well as several primary isolates, would enable these siRNAs to be used as potent antiviral tools for investigations with cells derived from the central nervous system in order to evaluate their therapeutic potential and assess their utility in inhibiting HIV-1 neuropathogenesis and neuroinvasion.

Antiviral Agents↗
PubMed2003

The N-terminal domain of APJ, a CNS-based coreceptor for HIV-1, is essential for its receptor function and coreceptor activity.

The human APJ, a G protein-coupled seven-transmembrane receptor, has been found to be dramatically expressed in the human central nervous system (CNS) and also to serve as a coreceptor for the entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). Studies with animal models suggested that APJ and its natural ligand, apelin, play an important role in the central control of body fluid homeostasis, and in regulation of blood pressure and cardiac contractility. In this study, we characterize the structural and functional determinants of the N-terminal domain of APJ in interactions with its natural ligand and HIV-1 envelope glycoprotein. We demonstrate that the second 10 residues of the N-terminal domain of APJ are critical for association with apelin, while the first 20 amino acids play an important role in supporting cell-cell fusion mediated by HIV-1 gp120. With site-directed mutagenesis, we have identified that the negatively charged amino acid residues Glu20 and Asp23 are involved in receptor and coreceptor functions, but residues Tyr10 and Tyr11 substantially contribute to coreceptor function for both T-tropic (CXCR4) and dual-tropic (CXCR4 and CCR5) HIV-1 isolates. Thus, this study provides potentially important information for further characterizing APJ-apelin functions in vitro and in vivo and designing small molecules for treatment of HIV-1 infection in the CNS.

Amino Acid Sequence↗
PubMed2003

Genotypic alteration of HAART-persistent HIV-1 reservoirs in vivo.

Three HIV-1-infected individuals, on virally-suppressive highly active anti-retroviral therapy (HAART), were treated in vivo with anti-retroviral inhibitor intensification and cell stimulatory therapies in attempting to eradicate latent viral reservoirs. Afterwards, the patients ceased all anti-retroviral drugs. Sequences of the V3 region of HIV-1 envelope protein (ENV) from patient peripheral blood mononuclear cell (PBMC) proviral DNA, patient blood plasma viral RNA and virion-associated RNA from viruses amplified by patient cell co-culture, were obtained before, during, and certain times after the clinical regimen. As anticipated, the V3 loop sequencing results indicate diversity in viral strain complexity among the individual patients. However, the detection of unique V3 ENV signature sequences or V3 signatures of low frequency, relative to those observed prior to therapy, indicate that the expression of specific viruses, or viruses of low abundance, can be induced through stimulation in vivo. Furthermore, this stimulation or general immune activation therapy (IAT) approach, consisting of administration of the anti-T-cell receptor antibody, OKT3, and IL-2 in vivo, appeared to have subsequently altered the genotype of the persistent viral reservoir in peripheral blood cells for two of the three patients.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo.

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain.

Animals↗
PubMed2003

Structural and functional study of the apelin-13 peptide, an endogenous ligand of the HIV-1 coreceptor, APJ.

The APJ receptor is widely expressed in the human central nervous system (CNS). Apelin was recently identified as the endogenous peptidic ligand for human APJ. Studies with animal models suggested that APJ and apelin play an important role in the hypothalamic regulation of water intake and the endocrine axis, in the regulation of blood pressure, and in cardiac contractility. Apelin has been found to block the activity of APJ as a human immunodeficiency virus type I (HIV-1) coreceptor. In this study, we combined chemical synthetic approaches with alanine substitution to evaluate the structural requirements for interactions with the APJ receptor. We demonstrated that apelin peptides in aqueous solution adopt a random conformation, and the positive charge and hydrophobic residues of apelin-13 play important roles in interactions with the APJ receptor. We have observed an important correlation between receptor binding affinity and cell-cell fusion inhibitory activity. The elucidation of structural requirements of apelin-13 in its interaction with the APJ receptor is critical for further investigation of apelin-APJ functions in vivo and in the design of small molecular inhibitors for potential treatment of HIV-1 infection in the CNS.

Alanine↗
PubMed2003

Binding of ALX40-4C to APJ, a CNS-based receptor, inhibits its utilization as a co-receptor by HIV-1.

APJ, a G protein-coupled seven-transmembrane receptor, has been shown to serve as a co-receptor for the entry of human immunodeficiency virus type 1 (HIV-1), and it is dramatically expressed in central nervous system (CNS)-based cells. ALX40-4C was identified as a small-molecule antagonist of the chemokine receptor CXCR4, which can specifically inhibit HIV-1 entry via this co-receptor. In this study, we demonstrated that ALX40-4C inhibited both APJ- and CXCR4/APJ-mediated cell membrane fusion in a dose-dependent manner. In competitive binding assays, (125)I-Apelin13 was replaced by ALX40-4C with an IC(50) of 2.9 microM, as compared with an IC(50) of 0.2 nM for Apelin13. Furthermore, ALX40-4C could block ligand-induced APJ internalization and signaling. ALX40-4C, as an antagonist to APJ, directly binds to and prevents use of APJ as a HIV-1 co-receptor. Thus, ALX-4C has potential utility for further elucidation of HIV-1 neuropathogenesis and therapy of HIV-1-induced encephalopathy.

Apelin Receptors↗
PubMed2003

Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA.

The leader RNA sequence of human immunodeficiency virus type 1 (HIV-1) consists of a complex series of stem loop structures that are critical for viral replication. Three-dimensional structural analysis by NMR of one of these structures, the SL1 stem loop of the packaging signal region, revealed a highly conserved purine rich loop with a structure nearly identical to the Rev-binding loop of the Rev response element. Using band-shift assays, surface plasmon resonance, and further NMR analysis, we demonstrate that this loop binds Rev. HIV-1 appears to have a second Rev-binding site close to the major splice donor site that may have an additional role in the viral life cycle.

5' Untranslated Regions↗
PubMed2003

Slower progression of HIV-1 infection in persons with GB virus C co-infection correlates with an intact T-helper 1 cytokine profile.

BACKGROUND: Progression to AIDS is slower in persons infected with both HIV-1 and GB virus C (GBV-C), also known as hepatitis G virus. OBJECTIVE: To compare clinical, virologic, and immunologic variables in HIV-1-seropositive patients with and without GBV-C co-infection. DESIGN: Subanalysis of a prospective cohort study. SETTING: Institute of Infectious Diseases, University of Catania, Catania, Italy. PATIENTS: 80 asymptomatic HIV-1-seropositive patients. MEASUREMENTS: GBV-C RNA level; plasma HIV-1 viral load; CD4(+) cell counts; and serum levels of interleukin (IL)-2, IL-4, IL-10, and IL-12. RESULTS: At the start of the study, plasma GBV-C RNA was detected in 17 patients (21%). During follow-up, IL-2 and IL-12 levels decreased significantly (P = 0.005 and P = 0.01, respectively) and IL-4 and IL-10 levels increased significantly (P = 0.01 and P = 0.004, respectively) in the GBV-C-negative group but did not change substantially in the GBV-C-positive group. Each measured variable differed significantly between GBV-C-positive and GBV-C-negative groups during follow-up (P < 0.001 for IL-12, IL-4, and IL-10; P = 0.002 for IL-2). CONCLUSION: GB virus C may immunologically interfere with progression of HIV-1 infection to AIDS by maintaining an intact T-helper 1 cytokine profile.

Acquired Immunodeficiency Syndrome↗
PubMed2003

The cytidine deaminase CEM15 induces hypermutation in newly synthesized HIV-1 DNA.

High mutation frequency during reverse transcription has a principal role in the genetic variation of primate lentiviral populations. It is the main driving force for the generation of drug resistance and the escape from immune surveillance. G to A hypermutation is one of the characteristics of primate lentiviruses, as well as other retroviruses, during replication in vivo and in cell culture. The molecular mechanisms of this process, however, remain to be clarified. Here, we demonstrate that CEM15 (also known as apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G; APOBEC3G), an endogenous inhibitor of human immunodeficiency virus type 1 (HIV-1) replication, is a cytidine deaminase and is able to induce G to A hypermutation in newly synthesized viral DNA. This effect can be counteracted by the HIV-1 virion infectivity factor (Vif). It seems that this viral DNA mutator is a viral defence mechanism in host cells that may induce either lethal hypermutation or instability of the incoming nascent viral reverse transcripts, which could account for the Vif-defective phenotype. Importantly, the accumulation of CEM15-mediated non-lethal hypermutation in the replicating viral genome could potently contribute to the genetic variation of primate lentiviral populations.

APOBEC-3G Deaminase↗
PubMed2003

Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ.

APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.

Animals↗
PubMed2003

Replication of lentiviruses.

Lentiviruses belong to a subfamily of the retroviruses usually associated with persistent infections in animals and humans. They have complex replication cycles involving numerous regulatory and accessory proteins, which sets them apart from the oncoretroviruses and spumaviruses, the two other main subfamilies of the retroviruses. Studies over the years have elucidated the various molecular mechanisms involved in the replication of lentiviruses. The first step involves the fusion of the envelope glycoprotein (gp120) to the host cell membrane followed by entry of the virus into the host cell. Immediately following viral entry is reverse transcription, integration, gene expression, encapsidation, budding and lastly virus maturation. This review focuses on the molecular mechanisms involved in the lentiviral replication, using human immunodeficiency virus type I (HIV-1) as an example.

Animals↗
PubMed2003

Twenty years of therapy for HIV-1 infection.

Antiretroviral therapy, where available, has transformed HIV-1 disease into a treatable and somewhat chronic infection. This article summarizes the accomplishments thus far and what lies ahead in our struggle to improve the treatment of, and possibly eliminate, HIV-1 infection.

AIDS Vaccines↗
PubMed2003

Peripheral blood Dendritic cells are not a major reservoir for HIV type 1 in infected individuals on virally suppressive HAART.

Dendritic cells (DCs) are potent antigen-presenting cells, and their physiological localization in tissues that interact with the external environment is important as a first barrier against pathogens such as human immunodeficiency virus type I (HIV-1). Several models have been proposed to explain the possible role of DCs as a reservoir for HIV-1 in patients on virally suppressive highly active antiretroviral therapy (HAART). However, the low yield of cell isolates has made this evaluation a difficult task. The present study analyzes whether peripheral blood DCs from HIV-1-infected individuals on virally suppressive HAART, with plasma HIV-1 RNA levels of less than 50 copies/ml, carry either HIV-1 provirus and/or HIV-1 virions. Peripheral blood DCs were isolated from a cohort of 10 HIV-1-seropositive men taking suppressive HAART. In five patients, plasmacytoid and myeloid dendritic cells were isolated to attempt to identify their respective roles in HIV-1 residual disease. Viral out-growth assays were performed in vitro, as well as gag and R/U5 polymerase chain reaction (PCR) amplification of viral RNA and DNA, respectively, from DC and peripheral blood mononuclear cell (PBMC) extracts. Fluorescence activated cell-sorting (FACS) data revealed cellular yields from 85.90 to 95.18%, of relatively pure DCs isolated from patients' PBMCs. Although HIV-1 RNA gag and DNA RU/5 were detected in all PBMC samples isolated from the patients, proviral DNA and viral RNA forms were not detected in any of the DC isolates. In addition, no replication-competent virus was demonstrated in DC coculture assays, while virus was isolated from each patients' CD8+ T-lymphocyte-depleted PBMC cocultures. Furthermore, HIV-1 gag proviral DNA was not detected in either plasmacytoid or myeloid DC subfractions. The current study suggests that in HIV-1-infected individuals treated with suppressive HAART, peripheral blood DCs do not carry HIV-1 proviral DNA or viral particles attached to their surface. These populations of peripheral blood DCs are likely not a major HIV-1 reservoir in patients on HAART with clinically undetectable plasma viral RNA.

Anti-HIV Agents↗
PubMed2003

Inhibition of HIV-1 in the central nervous system by IFN-alpha2 delivered by an SV40 vector.

In human immunodeficiency virus type 1 (HIV-1)-infected individuals, virus-induced production of interferon alpha (IFN-alpha) is impaired. In order to obtain regulated expression of IFN-alpha that responds to HIV-1 infection, a recombinant SV40 vector was designed that carries the human IFN-alpha2 cDNA under the control of the HIV-1 long terminal repeat (LTR) (SV[HIVLTR]IFN). Thus, the IFN-alpha2 gene would be trans-activated on infection with HIV-1. This vector was tested to determine if central nervous system (CNS) cell types that may be potential HIV-1 targets could be transduced and protected from HIV. SV[HIVLTR]IFN transduced NT2 cells, a human neuronal precursor cell line, mature neurons derived from NT2 precursor cells, and human primary monocyte-derived macrophages. IFN-alpha2 expression was retained in mature neurons after SV[HIVLTR]IFN-transduced NT2 precursor cells were induced to differentiate using retinoic acid. IFN-alpha expression was detected only after exposing transduced cells to HIV. Furthermore, SV[HIVLTR]IFN-delivered IFN-alpha2 expression significantly inhibited replication of multiple strains of HIV in both NT2 and NT2-derived mature neurons. SV[HIVLTR]IFN transduction also inhibited HIV-1(BaL) replication in human primary monocyte-derived macrophages. Therefore, we have demonstrated the effectiveness of IFN-alpha2, delivered by an SV40 vector driven by HIV-1 LTR as a promoter, to protect several CNS-based, potentially HIV-susceptible cell types. These findings may have implications for therapy of HIV-1 infection in the CNS.

Central Nervous System Diseases↗
PubMed2003

Treatment of Mycobacterium avium complex immune reconstitution disease in HIV-1-infected individuals.

Immune reconstitution disease caused by Mycobacterium avium complex (MAC) infection presenting shortly after the introduction of highly active antiretroviral therapy (HAART) has been reported with increasing frequency in persons with HIV-1 infection during the past several years. Several therapeutic modalities have been utilized for this entity, but the optimal means of treating MAC immune reconstitution disease remains unclear. We now describe a patient who underwent some of these therapies. We then review the therapeutic outcomes from the numerous case reports of this disorder. Finally, we propose recommendations and a clinical algorithm regarding the optimal means of treatment of MAC immune reconstitution disease during HIV-1 infection.

AIDS-Related Opportunistic Infections↗
PubMed2003

Second-generation rabies virus-based vaccine vectors expressing human immunodeficiency virus type 1 gag have greatly reduced pathogenicity but are highly immunogenic.

Rabies virus (RV) vaccine strain-based vectors show great promise as vaccines against other viral diseases such as human immunodeficiency virus type 1 (HIV-1) infection and hepatitis C, but a low residual pathogenicity remains a concern for their use. Here we describe several highly attenuated second-generation RV-based vaccine vehicles expressing HIV-1 Gag. For this approach, we modified the previously described RV vaccine vector SPBN by replacing the arginine at position 333 (R333) within the RV glycoprotein (G) with glutamic acid (E333), deleting 43 amino acids of the RV G cytoplasmic domain (CD), or combining the R333 exchange and the CD deletion. In addition, we constructed a new RV vector that expresses HIV-1 Gag from an RV transcription unit upstream of the RV phosphoprotein gene (BNSP-Gag) instead of upstream of the G gene. As expected and as demonstrated for SPBN-Gag, all vaccine vehicles were apathogenic after peripheral administration. However, the new, second-generation vaccine vectors containing modifications in the RV G were also apathogenic after intracranial infection with 10(5) infectious particles, and BNSP-Gag produced a 50%-reduced mortality in mice. Of note, the observed attenuation of pathogenicity did not result in either the attenuation of the humoral response against the RV G or the previously observed robust cellular response against HIV-1 Gag. These findings demonstrate that very safe and highly effective RV-based vaccines can be constructed and further emphasize their potential utility as efficacious antiviral vaccines.

AIDS Vaccines↗