Inhibition Of Human Immunodeficiency Virus Replication Research Articles

369. Selective Expansion of Human T Lymphocytes Transduced by HIV Vectors Containing IMPDH2 and Anti-HIV siRNA Genes

To achieve effective protection of T cells from HIV infection using gene therapy approaches, a sufficient number of T cells expressing anti-HIV gene(s) are required. However, vectors derived from Moloney murine leukemia virus or human immunodeficiency virus (HIV) are relatively inefficient at delivering foreign genes into hematopoietic stem cells (HSCs) from which the genetically modified T cells could be derived. In this study, we evaluated a mutated human type II inosine-5′ monophosphate dehydrogenase (IMPDH2) for its ability to confer cell resistance to drugs such as mycophenolic acid (MPA) and its precursor mycophenolate mofetil (MMF) (Serrano et al. Mol Therapy 3:S152, 2001). The safety profile of these drugs in clinics may allow direct selection and expansion of the transduced T cell in vivo. The cDNA for the mutant IMPDH2, with two amino acid substitutions, was placed under the control of the promoter of Rous sarcoma virus (RSV) and inserted into pHIV7, a 3rd generation HIV vector containing the GFP gene. H9 cells were transduced with the IMPDH2 vector and selected in the following escalating doses of MPA: 3, 6, 12, 18 and 24 × 10−6 M for 24 days. The viability at these doses was 94, 94, 71, 51 and 12%, respectively, whereas cells transduced with a control GFP vector were eliminated at all doses. The percentage of GFP increased from 5.2% before drug selection to 90–97% after drug selection, suggesting that IMPDH2 could serve as an effective selection marker for the transduced cells. To treat HIV infection, we evaluated a small interfering RNA (siRNA) specific against the p24 coding region for its ability to down-regulate HIV replication. A hairpin p24 siRNA gene was placed under the control of the H1 promoter and inserted into pHIV7. H9 cells were transduced with this vector and challenged with NL4-3 at different doses. Our results showed an effective inhibition of HIV replication, from 2 to 6 log reduction, by the p24 siRNA for up to 30 days after challenge in culture. The IMPDH2 cDNA was then inserted into the vector containing the siRNA gene and the effect of drug selection on protection of the transduced cells from HIV infection is under investigation.

Release of anti-HIV mediators after administration of leukotriene B4 to humans.

CD8(+) T cells can control human immunodeficiency virus (HIV) through the lysis of infected cells and the release of soluble mediators, such as macrophage inflammatory protein (MIP)-1 beta, which prevent entry of HIV and/or inhibit HIV replication. Because neutrophils represent a major source of alpha-defensins and, to a lesser extent, MIP-1 beta, we determined whether leukotriene B(4) (LTB(4)), a potent neutrophil agonist, would trigger the release of these 2 anti-HIV peptides. Plasma samples from HIV-uninfected subjects receiving intravenous bolus of LTB(4) were analyzed for alpha-defensins and MIP-1 beta levels by use of enzyme-linked immunosorbent assay. Furthermore, in vitro analysis of intracellular and secreted levels of alpha-defensins of resting and LTB(4)-activated neutrophils from HIV-uninfected and HIV-infected subjects were determined. LTB(4) modulation of CD63 and CD66b markers associated with degranulation were studied by use of flow cytometry. Chemotaxis of neutrophils from HIV-uninfected and HIV-infected subjects toward LTB(4) or interleukin (IL)-8 was determined by use of migration assays. Administration of LTB(4) to humans caused a dose-dependent plasmatic increase in alpha-defensins and MIP-1 beta proteins, with peak levels observed 2 h after administration of LTB(4). Neutrophils isolated from HIV-infected and HIV-uninfected subjects contained similar levels of stored alpha-defensins that were effectively secreted in vitro, in response to LTB(4) activation. Chemotaxis of neutrophils toward LTB(4) or IL-8 was identical among the groups of subjects. LTB(4) induced the secretion alpha-defensins and MIP-1 beta. Neutrophils from HIV-infected subjects were fully responsive to LTB(4), which highlights a potential usefulness of this lipid mediator in the management of HIV infection.

Gene therapy for HIV/AIDS: the potential for a new therapeutic regimen.

Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). HIV/AIDS is a disease that, compared with the not so distant past, is now better held in check by current antiretroviral drugs. However, it remains a disease not solved. Highly active antiretroviral therapy (HAART) generally uses two non-nucleoside and one nucleoside reverse transcriptase (RT) inhibitor or two non-nucleoside RT and one protease inhibitor. HAART is far more effective than the mono- or duo-therapy of the past, which used compounds like the nucleoside reverse transcriptase inhibitor AZT or two nucleoside reverse transcriptase inhibitors. However, even with the relatively potent drug cocktails that comprise HAART, there are the issues of (i). HIV escape mutants, (ii). an apparent need to take the drugs in an ongoing manner, and (iii). the drugs' side effects that are often severe. This review speaks to the potential addition to these potent regimens of another regimen, namely the genetic modification of target hematopoietic cells. Such a new treatment paradigm is conceptually attractive as it may yield the constant intracellular expression of an anti-HIV gene that acts to inhibit HIV replication and pathogenicity. A body of preclinical work exists showing the inhibition of HIV replication and decreased HIV pathogenicity by anti-HIV genetic agents. This preclinical work used hematopoietic cell lines and primary cells as the target tissue. More recently, several clinical trials have sought to test this concept in vivo.

Inhibition of HIV-1 replication by anti-trans-activation responsive polyamide nucleotide analog

Efficient replication and gene expression of human immunodeficiency virus-1 (HIV-1) involves specific interaction of the viral protein Tat, with its trans-activation responsive element (TAR) which forms a highly stable stem-loop structure. We have earlier shown that a 15-mer polyamide nucleotide analog (PNA) targeted to the loop and bulge region of TAR blocks Tat-mediated transactivation of the HIV-1 LTR both in vitro and in cell culture (Mayhood et al., Biochemistry 39 (2000) 11532). In this communication, we have designed four anti-TAR PNAs of different length such that they either complement the entire loop and bulge region (PNA TAR-16 and PNA TAR-15) or are short of few sequences in the loop (PNA TAR-13) or in both the loop and bulge (PNA TAR-12), and examined their functional efficacy in vitro as well as in HIV-1 infected cell cultures. All four anti-TAR PNAs showed strong affinity for TAR RNA, while their ability to block in vitro reverse transcription was influenced by their length. In marked contrast to PNA TAR-12 and PNA TAR-13, the two longer PNA TARs were able to efficiently sequester the targeted site on TAR RNA, thereby substantially inhibiting Tat-mediated transactivation of the HIV-1 LTR. Further, a substantial inhibition of virus production was noted with all the four anti-TAR PNA, with PNA TAR-16 exhibiting a dramatic reduction of HIV-1 production by nearly 99%. These results point to PNA TAR-16 as a potential anti-HIV agent.

Acute human immunodeficiency virus replication causes a rapid and persistent impairment of Vgamma9Vdelta2 T cells in chronically infected patients undergoing structured treatment interruption.

T cells expressing Vgamma9Vdelta2 display lytic and proliferative responses against human immunodeficiency virus (HIV)-infected cells and release antiviral soluble factors. Chronic HIV-positive patients have deep changes in the composition and function of the circulating gammadelta T cell pool that are restored by highly active antiretroviral therapy (HAART). gammadelta T cells were analyzed during the rapid plasma HIV RNA rebound in HIV-infected patients undergoing structured treatment interruption (STI). A loss in circulating Vgamma9Vdelta2 T cells was observed, indicating that acute HIV replication may influence Vgamma9Vdelta2 homeostasis. These cells were lost among CD45RA(-)CD27(-) Vgamma9Vdelta2 T cell effectors, and a significant unresponsiveness, measured as antigen-driven interferon-gamma production, was observed during STI. After HAART resumption and consequent inhibition of HIV replication, Vgamma9Vdelta2 T cell reactivity was restored both quantitatively and functionally. These observations indicate that Vgamma9Vdelta2 T cells are activated early after active HIV replication but are rapidly lost when viremia is not controlled.

Is antisense an appropriate nomenclature or design for oligodeoxynucleotides aimed at the inhibition of HIV-1 replication?

We have evaluated the specificity and the variation in activity against human immunodeficiency virus (HIV) infection of antisense oligodeoxynucleotides (ODNs) with regard to factors such as dose-response range, number and choice of experimental controls, backbone modifications of the ODNs, type of cell infection, length of assays, and delivery approach. The highest level of inhibition was achieved in our long-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB0 and treated with free phosphorothioate-modified ODNs (PS-ODNs). The highest level of specificity was observed in our short-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free PS-ODNs. the highest potency (IC50 level) was observed in our short-term chronic-infection model with (DLS)-delivered ODNs in which the DLS delivery improved the ODN activity up to 106 times compared to the activity of free ODNs. Thus, the near blocking of HIV replication obtained when using PS-ODNs appears because of the addition of extracellular and/or membranar effects. The higher efficacy of PS-ODNs compared to unmodified ODNs, when both are delivered with the DLS system, was demonstrated solely in our short-term assay with MOLT-3 cells. Important variations in the level of sequence specificity were observed and depended on the type of control used and the type of cell assay employed. It seems that all 3 groups of control-tested, random, sense sequence, and non-antisense T30177 ODNs might have distinct activity and, consequently, different modes of action in inhibiting HIV replication. Our data buttress the notion that the contribution of the sequence-specific mediated mode of action is minor compared to the other mechanisms involved in ODN antiviral activity.

Inhibition of bacterial RNase P by aminoglycoside–arginine conjugates

The potential of RNAs and RNA–protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA ( trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense, decoy, and sense RNAs, and trans-dominant negative mutant Gag and Env proteins.

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense decoy and sense RNAS and trans-dominant negative mutant gag and env proteins

Oncoretroviral vectors were engineered to allow constitutive expression of an antisense RNA and the trans-activator of transcription (Tat)-inducible expression of a mRNA containing the trans-activation response (TAR) element, the Rev response element (RRE), and the efficient packaging signal (Psi(e) of human immunodeficiency virus-1 (HIV-1) RNA. Nuclear export of this mRNA by the regulator of expression of virion proteins (Rev) would allow its translation into wild type (WT) (MoTN-Ti-GE-Ri- Ter) or trans-dominant negative mutant (TDM) (MoTN-Ti-GmEm-Ri-Ter) Gag and Env proteins. Thus, the antisense RNA produced in a constitutive manner would ensure that even if there is leaky expression, no WT/TDM Gag or Env protein would be produced in the uninfected cells. If cells become infected by HIV-1, the antisense RNA would inhibit HIV-1 replication. Failure on the part of antisense RNA to inhibit virus replication would allow GE/GmEm mRNA production. The GE/GmEm mRNA would cause partial inhibition of HIV-1 replication as it contains the TAR, RRE, and Psi(e) signal sequences. Translation of GmEm mRNA would give rise to TDM Gag and Env proteins, which would further decrease progeny virus infectivity. Tat- and Rev-inducibility was demonstrated in transfected HeLa and HeLa-Tev cells. Full-length WT/TDM Gag production was confirmed by Western blot analysis. Amphotropic vector particles were used to transduce a human CD4+ T-lymphoid cell line, and the stable transductants were challenged with HIV-1. Virus replication was better inhibited by the MoTN-Ti-GE-Ri-Ter vector than by the MoTN-Ti-GmEm-Ri-Ter vector. Inhibition of HIV-1 replication was also demonstrated in transduced CD4+ human peripheral blood T lymphocytes (PBLs). Moreover, our results suggest that cloning in the reverse transcriptional orientation must be avoided to prevent antisense RNA-mediated inhibition of transgene and endogenous gene expression.

Recent studies on natural products as anti-HIV agents.

The discovery of medicinal agents capable of specifically inhibiting human immunodeficiency virus (HIV) is urgently needed due to its globally widespread infection. Most of clinically useful anti-HIV agents are nucleosides but their use is limited due to their severe toxicity and emerging drug resistance. More than 50% of world marketed drugs have their origin of the nature. Natural products, of which structural diversity is so broad, are good sources for the effective discovery of anti-HIV agents with decreased toxicity. Over the past decade, substantial progress has been made in research on the natural products for the anti-HIV agents. New natural products that have potent anti-HIV activities with novel structures were reviewed in this article. These compounds, isolated mainly from medicinal plants, in this review have been classified as secondary metabolites such as terpenes, phenolics, and naturally scarce peptides and sugars. Especially, terpenes and phenol substances have gained much interest due to their significant anti-HIV activities along with their structural diversity. Recent studies also showed that several polysaccharides are effective inhibitors of HIV replication. Most of chemotherapeutic targets reviewed in this article are found to be HIV reverse transcriptase (RT).

CD8(+)-Cell antiviral factor activity is not restricted to human immunodeficiency virus (HIV)-specific T cells and can block HIV replication after initiation of reverse transcription.

CD8(+) lymphocytes from human immunodeficiency virus (HIV)-infected patients can suppress in vitro HIV replication in CD4(+) T cells by a noncytolytic mechanism involving secreted CD8(+)-cell antiviral factor(s) (CAF). Using an HIV Nef-specific cytotoxic-T-lymphocyte (CTL) line and autologous CD4(+) T cells infected with a nef-deleted HIV-1 virus, we demonstrated that, after a priming antigenic stimulation, this suppression does not require the presence of the specific antigen during the effector phase. Furthermore, using an Epstein-Barr virus (EBV)-specific CTL line from an HIV-seronegative donor, we demonstrated that the ability to inhibit HIV replication in a noncytolytic manner is not restricted to HIV-specific effector cells; indeed, EBV-specific CTL were as efficient as HIV-specific effectors in suppressing R5 or X4 HIV-1 strain replication in vitro. This HIV-suppressive activity mediated by a soluble factor(s) present in the culture supernatant was detectable for up to 14 days following stimulation of EBV-specific CD8(+) cells with the cognate epitope peptide. Following acute infection of CEM cells with an X4 strain of HIV-1, EBV-specific CTL line supernatant containing HIV-suppressive activity did not block virus entry but was shown to interfere with virus replication after the first template switching of reverse transcription. Our results suggest that the noncytolytic control of HIV replication by EBV-specific CD8(+) T lymphocytes corresponded to a CAF-like activity and thus demonstrate that CAF production may not be restricted to CTL induced during HIV disease. Moreover, CAF acts after reverse transcription at least for X4 isolate replication inhibition.

The Use of Ribozyme Gene Therapy for the Inhibition of HIV Replication and its Pathogenic Sequelae

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.

Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance

AbstractHuman immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4+ and CXCR4−, were observed: morphological analysis of the sorted populations showed that the CXCR4+ cells were largely eosinophils and the CXCR4− were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, ≈30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on ≈15% of the starting CD34+ HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4− G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1α [MIP-1α], MIP-1β and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.

Lineage-Specific Expression of Human Immunodeficiency Virus (HIV) Receptor/Coreceptors in Differentiating Hematopoietic Precursors: Correlation With Susceptibility to T- and M-Tropic HIV and Chemokine-Mediated HIV Resistance

Human immunodeficiency virus (HIV) entry is mediated not only by the CD4 receptor, but also by interaction with closely related molecules that act as membrane coreceptors. We have analyzed mRNA expression and/or cell membrane exposition of the coreceptors most widely used by diverse HIV-1 strains (CXCR4, CCR5, and CCR3) on purified hematopoietic progenitor cells (HPCs) induced in liquid suspension culture to unilineage differentiation/maturation through the erythroid (E), granulocytic (G), megakaryocytic (Mk), and monocytic (Mo) lineages. Reverse transcriptase-polymerase chain reaction (RT-PCR) and cytofluorimetric analysis showed the presence of both CXCR4 and CCR5 in quiescent HPCs, but failed to detect CCR3-specific transcripts. Chemokine expression in HPC progenies showed that CXCR4 receptor is detected on the majority of MKs from early to late stages of maturation, whereas it is moderately decreased in the Mo lineage. In the G pathway, two distinct cell populations, CXCR4(+) and CXCR4(-), were observed: morphological analysis of the sorted populations showed that the CXCR4(+) cells were largely eosinophils and the CXCR4(-) were granulocytes of the neutrophilic series. Furthermore, in the E pathway, CXCR4 was almost completely absent. CCR5 expression is restricted to Mo cultures, ie, approximately 30% to 80% cells throughout all monocytopoietic differentiation/maturation stages. Finally, CCR3 mRNA is always absent in all the unilineage cultures. Evaluation of CD4 expression by flow cytometry on both quiescent HPCs and differentiating unilineage precursors showed that the CD4 receptor is present on approximately 15% of the starting CD34(+) HPC population, highly expressed in the Mo lineage up to 80% at terminal maturation, present on 20% to 30% of maturing Mks, and not detectable in either the E or G lineage. Expression of CD4 receptor together with CXCR4 and/or CCR5 coreceptor in the four lineages correlates with hematopoietic precursor susceptibility to T-lymphotropic and macrophage (M)-tropic HIV strains infection: (1) CD4(-) G and E cells were resistant to both M-tropic and T-lymphotropic strains; (2) HPC-derived Mks were susceptible to T-tropic, but resistant to M-tropic, infection; (3) Mo differentiating cells efficiently replicate both HIV strains. Furthermore, we showed that the CXCR4 and CCR5 ligands (stromal-derived factor 1 and macrophage-inflammatory protein-1alpha [MIP-1alpha], MIP-1beta and RANTES, respectively) inhibit HIV replication in both maturing Mo and Mk cells. Taken together, our data show a lineage-specific modulation of chemokine receptor/coreceptor during hematopoietic cell differentiation and extend previous observations on the relationship between the expression of HIV receptor/coreceptors, susceptibility, and chemokine-mediated resistance to HIV infection.

Anti-human immunodeficiency virus type 1 (HIV-1) activity of 2'-fluoro-2',3'-dideoxyarabinosyladenine (F-ddA) used in combination with other mechanistically diverse inhibitors of HIV-1 replication.

2'-Fluoro-2'3'-dideoxyarabinosyladenine (F-ddA), a nucleoside reverse transcriptase inhibitor of human immunodeficiency virus (HIV) replication, is currently being evaluated in clinical trials. Future monotherapy for the treatment of HIV is unlikely owing to the rapid emergence of drug-resistant viruses, so F-ddA was evaluated in combination with a variety of mechanistically diverse inhibitors of HIV replication. Such in vitro studies provide insights into whether certain drug combinations yield synergistic antiviral activity or, more importantly, antagonistic antiviral activity or synergistic cytotoxicity. F-ddA exhibited synergistic antiviral interactions with representatives of each of the major classes of anti-HIV compounds, including other nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitors. Greatest levels of synergistic interaction were detected when F-ddA was used in combination with the non-nucleoside compounds nevirapine and costatolide, the nucleoside analogues and costatolide, the nucleoside analogues AZT, ddC and 3TC and the protease inhibitors ritonavir and nelfinavir. No evidence of either combination toxicity or antagonistic antiviral activity was detected with any of the tested compounds.

Human Immunodeficiency Virus-1 Infection Requires Pertussis Toxin Sensitive G-Protein-Coupled Signalling and Mediates cAMP Downregulation

The human immunodeficiency virus-1 (HIV-1) utilises CD4 and certain β-chemokine receptors, mainly CCR-5 and CXCR4, for attachment and virus entry into T-lymphocytes and monocytes/macrophages. CD4 and β-chemokine receptors participate in intracellular signalling via protein tyrosine kinases and G-protein-coupled signalling. The factors which influence HIV-1 replication and the intracellular signalling mechanisms elicited by the virus are not well understood. In this study, it was demonstrated that exposure of peripheral blood lymphocytes (PBLs) to a T-cell tropic strain of HIV-1 evokes signal(s) which results in downregulation of intracellular cAMP. In addition, pre-incubation of PBLs with the Gi-protein inhibitor Pertussis toxin mediated a significant inhibition of HIV-1 replication. These data strongly suggest that HIV-1 employs CD4 receptors and Gi-coupled proteins for entry into target cells and that productive HIV-1 infection is dependent on an active signalling event.

Gene Therapy for HIV Infections: Intracellular Immunization

Despite significant advances in the treatment of human immunodeficiency virus (HIV) infection in the past 10 years, it remains an incurable disease. The inability of traditional drug-based therapies to inhibit HIV replication effectively for extended periods of time has stimulated intense research to develop novel approaches for this disease. Current understanding of HIV molecular biology and pathogenesis has opened the way for the development of gene therapy strategies for HIV infections. In this context, a number of intracellular immunization-based strategies have been evaluated, and some of them have reached the stage of phase I/II human clinical trials. These strategies include the use of single-chain antibodies, capsid-targeted viral inactivation, transdominant negative mutants, ribozymes, antisense oligonucleotides and RNA decoys. While a number of issues remain to be studied before intracellular immunization can be applied to the treatment of HIV infections, the significant progress already made in this field is likely to lead to clinical applications.

Oltipraz, a novel inhibitor of hepatitis B virus transcription through elevation of p53 protein.

Molecular epidemiological studies of populations at high risk for liver cancer have shown that hepatitis B virus (HBV) and aflatoxin B1 exposures are two major risk factors for this disease. Oltipraz is currently being considered for clinical trial to protect against aflatoxin B1-induced hepatocarcinogenesis based on its proven protective effect in many different animal models. In addition, oltipraz inhibits human immunodeficiency virus (HIV) replication. The inactivation of reverse transcriptase of HIV appears to be the antiviral mechanism. It has been demonstrated that a number of compounds that inhibit HIV replication also inhibit HBV replication in vitro. Therefore, we tested the possibility of oltipraz blocking HBV replication in 2.2.15 cells (clonal cells derived from HepG2 cells that were transfected with a plasmid containing HBV DNA) in vitro. Results of the experiments indicate that oltipraz has a dose-dependent inhibitory effect on HBV replication and specifically blocks HBV transcription in 2.2.15 cells. In addition, oltipraz induces endogenous wild-type p53 protein in a dose- and time-course-dependent manner. Taken together, we speculate that the effects of oltipraz against replication of HBV and specific blocking of HBV transcription may be through the induction of p53-mediated pathway in 2.2.15 cells. In addition to its known chemopreventive action on aflatoxin B1 hepatocarcinogenesis, oltipraz was shown here to inhibit HBV replication. These dual effects put oltipraz as the excellent candidate for the chemopreventive agent of human hepatocellular carcinoma.

Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors.

We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39-48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56(lck) (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56(lck) or NF-kappaB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56(lck) signaling, and may involve a critical region for CD4 dimerization.

3'-End modification of the adenoviral VA1 gene affects its expression in human cells: consequences for the design of chimeric VA1 RNA ribozymes.

Polymerase III (pol III)-dependent genes, like the adenoviral VA1 gene, are of particular interest for expressing small therapeutic RNAs into cells. A new VA1 RNA carrier molecule was generated through the deletion of the VA1 RNA central domain to give rise to the VAdeltaIV RNA vector that was devoid of undesirable physiologic activity (i.e., inhibition of the interferon-induced protein kinase, PKR). This vector was used to express in human cells hammerhead ribozymes targeted against the human immunodeficiency virus (HIV). Eight anti-HIV ribozymes were inserted at the 3'-end of this vector immediately before the four T-residues that serve as a transcription termination signal. Although the constructs were active in vitro, they failed to inhibit HIV replication in transient assays. Analysis of the intracellular ribozyme expression in cells revealed several anomalies. First, using mutant derivatives, we showed that the presence of two or three consecutive T-residues in the ribozyme portion was sufficient to promote the release of anomalous short transcripts. Second, when the ribozyme did not contain T-rich sequence, full-length transcripts were produced, but these transcripts were very unstable and were retained in the cell nucleus. In contrast, insertion of the ribozyme in place of the central domain of VA1 RNA led to production of full-length transcripts that were stable and located in the cytoplasm but that were not found to be active in vitro. Taken together, these results have important consequences for the future design of active intracellular ribozymes based on the use of pol III-transcribed genes.