Human Immunodeficiency Virus Type 1 Reactivation Research Articles

Hepatitis C Therapy With Interferon-α and Ribavirin Reduces CD4 T-Cell–Associated HIV-1 DNA in HIV-1/Hepatitis C Virus–Coinfected Patients

Combined treatment with interferon alpha (IFN-α) and ribavirin (RBV) can effectively cure HCV infection in a significant proportion of patients, but effects of this regimen on cellular reservoirs for human immunodeficiency virus type 1 (HIV-1) are unknown. Here, we show that treatment with IFN-α/RBV led to a moderate but significant and sustained decline of HIV-1 DNA in CD4 T cells from HIV-1/hepatitis C virus-coinfected patients receiving highly active antiretroviral therapy (n = 12). However, in vitro experiments failed to demonstrate an effect of pharmacological doses of IFN-α on HIV-1 reactivation. Together, these data suggest that treatment with IFN-α/RBV can moderately reduce the reservoir of HIV-1-infected CD4 T cells that persists despite suppressive antiretroviral therapy.

Cycloviolacin O2 (CyO2) suppresses productive infection and augments the antiviral efficacy of nelfinavir in HIV‐1 infected monocytic cells

Human immunodeficiency virus type-1 (HIV-1), the etiologic agent of acquired immune deficiency syndrome (AIDS), is a global pandemic causing millions of deaths annually. Highly active antiretroviral therapy (HAART) greatly enhances lifespan but eventually causes debilitating side effects, in part, due to their chronic administration required to suppress HIV-1 replication. If treatment is discontinued, viral suppression is lost and dormant replication-competent monocytic cell reservoirs become reactivated, leading to viral recrudescence and progression to AIDS. Therefore, novel strategies to circumvent obstacles to HIV-1 therapy are critically needed. We evaluated the potentially therapeutic effects of cycloviolacin O2 (CyO2) on cell viability (MTT assay), membrane disruption (SYTOX Green uptake), p24 production [enzyme-linked immunosorbent assays (ELISA)], and proviral integration (PCR amplification) in U1 cells; a monocytic cell model of HIV-1 latency and reactivation. We demonstrate, for the first time, that CyO2 (0.5-5.0 μM) kills productively infected cells. Sub-toxic concentrations (<0.5 μM) of CyO2 disrupted plasma membranes in both latently-infected and productively-infected U1 cells and enhanced the antiviral efficacy of nelfinavir, a HIV-1 protease inhibitor (HPI). Interestingly, CyO2 also decreased virus production by activated U1 cells; however, this effect was not due to suppression of integrated provirus in U1 cells. This suggested that, in addition to the known pore-forming ability of cyclotides, a novel mode of antiviral activity may exist for CyO2. Our data indicate that CyO2 may be a promising candidate for the targeting HIV-1 reservoirs in monocytes, and their inclusion in adjuvant therapy approaches may augment the efficacy of HPIs and ultimately facilitate virus elimination.

Histone Deactylase Inhibitor SAHA Induces a Synergistic HIV-1 Reactivation by 12-O-Tetradecanoylphorbol-13-Acetate in Latently Infected Cells

Objectives: Recent studies have reported that human immunodeficiency virus type 1 (HIV-1) proviruses are strongly suppressed in the unique epigenetic environments caused by chromatin modifications such as acetylation and methylation. Therefore, optimized therapeutic strategies directed against the virus reservoir using these epigenetic modifying agents (EMAs) should cure HIV infection. Methods: Cytotoxicity and HIV-1 reactivation were determined using the PrestoBlue™ Cell Viability Reagent and p24 HIV ELISA, respectively. Results: EMAs, including histone deacetylase inhibitors (VPA and SAHA), DNA methyltransferase inhibitor (5′-Aza-CdR), histone methyltransferase inhibitor (ADOX) and 12-O-tetradecanoylphorbol-13-acetate (TPA), were used to reactivate proviruses in HIV-1 latently infected cells. The effect of monotreatment with these EMAs on HIV-1 reactivation was VPA or SAHA > 5′-Aza-CdR > ADOX. Even though cotreatment with these potential HIV-1 reactivating agents did not show any significant reactivation effects in HIV-1 latently infected cells, employing SAHA under TPA treatment demonstrated a dramatic synergistic effect on purging HIV-1 proviruses in all HIV-1 latently infected cells via the ERK and AP-1 pathways. Conclusions: These results suggest that the combined approaches of EMAs, cotreatment of SAHA and TPA, could provide an effective way to lead a decline of HIV-1 reservoirs in patients.

An &lt;em&gt;In vitro&lt;/em&gt; Co-infection Model to Study &lt;em&gt;Plasmodium falciparum&lt;/em&gt;-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission. The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed. Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An &lt;em&gt;In vitro&lt;/em&gt; Co-infection Model to Study &lt;em&gt;Plasmodium falciparum&lt;/em&gt;-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

Microbial Interaction of Periodontopathic Bacterium Porphyromonas gingivalis and HIV-Possible Causal Link of Periodontal Diseases to AIDS Progression-

A wide variety of infections, including bacteria, viruses, fungi and protozoa occur in the immunocompromised condition associated with human immunodeficiency virus type 1 (HIV-1) infection and acquired immunodeficiency syndrome (AIDS). Although these opportunistic infections are believed to arise as an effect of the immunodeficiency, these microbes sometimes promote the disease progression of HIV-1 infection by enhancing viral replication or modulating host immune responses. Here we review the experimental and clinical evidence supporting such causal relationships associated with periodontogenic bacteria. Periodontal disease, caused by subgingival infection with oral anaerobic bacteria, typically Porphyromonas gingivalis (P. gingivalis) belonging to the phylum Bacteroidetes, is found worldwide and is one of the most prevalent microbial diseases of mankind. Emerging evidence implicates the involvement of P. gingivalis infection in the progression of HIV-1 infection. We demonstrate that P. gingivalis can induce HIV-1 reactivation via chromatin modification, and that the bacterial metabolite butyric acid produced in anaerobic conditions is responsible for this effect. These findings suggest that periodontal diseases could act as a risk factor for HIV-1 reactivation in infected individuals and might contribute to AIDS progression. Furthermore, it would imply that prevention and early treatment of periodontitis involving P. gingivalis infection could effectively block further clinical progression of AIDS.

TRAF activation of C/EBPβ (NF-IL6) via p38 MAPK induces HIV-1 gene expression in monocytes/macrophages

C/EBPβ plays a pivotal role in activation of human immunodeficiency virus type 1 (HIV-1) in monocytes/macrophages. However, mechanisms for functional regulation of C/EBPβ remain uncharacterized. Previous studies indicated that NF-κB activation by tumor necrosis factor (TNF) receptor family, which activates TNF receptor associated factor (TRAF), induces HIV-1 expression. We found that TRAF signals activate HIV-1 LTR with mutations of NF-κB sites in promonocytic cell line U937, suggesting existence of an alternative HIV-1 activating pathway. In this study, we have characterized the signal transduction pathway of TRAF other than that leading to NF-κB, using U937 cell line, and its subline, U1, which is chronically infected by HIV-1. We show that signals downstream of TRAF2 and TRAF5 activate p38 MAPK, which directly phosphorylates C/EBPβ, and that activation of p38 MAPK potently activates C/EBPβ-mediated induction of HIV-1 gene expression. We also show TRAF2 and TRAF5 are expressed in monocytes/macrophages of spleen samples from HIV-1 infected patients. Identification of TRAF-p38 MAPK-CEBPβ pathway provides a new target for controlling reactivation of latent HIV-1 in monocytes/macrophages.

H. saimiri tyrosine-kinase interacting protein inhibits Tat function: A prototypic strategy for restricting HIV-1-induced cytopathic effects in immune cells

Herpesvirus saimiri (HVS)-transformed human T cells become permissive for X4 and R5 strains of human immunodeficiency virus type 1 (HIV-1), evidence that HVS-encoded proteins associated with T cell transformation enhance HIV-1 replication. Analyzing the contribution of transformation-associated bicistronic HVS open reading frames (ORF) to HIV-1 replication revealed expression of the second ORF saimiri transformation-associated protein type C (StpC) conferred the permissive phenotype to T cells. In contrast, expression of the first HVS ORF tyrosine-kinase interacting protein (Tip) in the absence of StpC enhanced restriction of HIV-1 replication in T cell lines and peripheral blood mononuclear cells. Understanding the mechanism whereby Tip enhanced restriction of HIV-1 replication may uncover unique pathways that could be targeted therapeutically. Here we report that Tip restricts HIV-1 replication in a monocyte-derived cell line and restricts reactivation of replication of HIV-1 in a T cell line harboring provirus. In this report, we begin to unravel the molecular underpinnings of Tip-mediated restriction. Tip mediates both lymphocyte-cell-specific kinase (Lck)-dependent and -independent effects on HIV-1 replication. We also provide evidence that Tip-mediated restriction is in part due to inhibition of Tat transactivation of the HIV-1 long terminal repeat (LTR). Expression of Tip in T cells increased activation of Stat1 and Stat3, as well as activation of protein kinase RNA-dependent (PKR/p68) and interferon-γ production. Taken together, these results provide evidence that Tip restricts HIV-1 replication and reactivation by inhibiting HIV-1 transcription while inducing an intercellular antiviral state. We propose that genetically engineered vectors driving Tip expression could provide a prototypic strategy for restricting HIV-1 replication and reactivation in diverse cell lineages.

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency.

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.

Diversity of acetylation targets and roles in transcriptional regulation: the human immunodeficiency virus type 1 promoter as a model system

Persuasive evidence has accumulated that reversible acetylation of proteins is key post-translational modification regulating transcription in eukaryotes. Deacetylase inhibitors (such as trichostatin A) modulate the expression of ∼2% of all cellular genes. We and others have demonstrated a marked transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) promoter in response to deacetylase inhibitors. Deacetylation events seem to be an important mechanism of HIV-1 transcriptional repression during latency, whereas acetylation events play critical functional roles in HIV-1 reactivation from latency. These deacetylation/acetylation events are implicated in chromatin remodeling of the viral promoter region, as well as in modulating the functional properties of cellular and viral transcription factors binding to this promoter region. Thereby, the HIV-1 promoter constitutes a unique regulatory model system to study the complex relationship between acetylation processes and transcriptional activity.

Induction of TAK (cyclin T1/P-TEFb) in purified resting CD4(+) T lymphocytes by combination of cytokines.

Combinations of cytokines are known to reactivate transcription and replication of latent human immunodeficiency virus type 1 (HIV-1) proviruses in resting CD4(+) T lymphocytes isolated from infected individuals. Transcription of the HIV-1 provirus by RNA polymerase II is strongly stimulated by the viral Tat protein. Tat function is mediated by a cellular protein kinase known as TAK (cyclin T1/P-TEFb) that is composed of Cdk9 and cyclin T1. We have found that treatment of peripheral blood lymphocytes and purified resting CD4(+) T lymphocytes with the combination of interleukin-2 (IL-2), IL-6, and tumor necrosis factor alpha resulted in an increase in Cdk9 and cyclin T1 protein levels and an increase in TAK enzymatic activity. The cytokine induction of TAK in resting CD4(+) T lymphocytes did not appear to require proliferation of lymphocytes. These results suggest that induction of TAK by cytokines secreted in the microenvironment of lymphoid tissue may be involved in the reactivation of HIV-1 in CD4(+) T lymphocytes harboring a latent provirus.

Phenylarsine oxide inhibits ex vivo HIV-1 expression

Phenylarsine oxide (PAO), which is described as an inhibitor of tyrosine phosphatase activity, inhibits H2O2 release from human peripheral blood mononuclear cells (PBMCs) as measured by electrochemistry. Since human immunodeficiency virus type 1 (HIV-1) replication is known to be favored under oxidative stress conditions, ex vivo experiments using uninfected PBMCs, primary monocytes or a latently infected promonocytic U1 cell line show that HIV-1 replication and reactivation, monitored by p24 antigen measurement, are inhibited by PAO in a time- and concentration-dependent manner. These observations can be linked with the inhibition of NF-kappa B activation when uninfected monocytes are induced by either tumor necrosis factor alpha (TNF-alpha) phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS).

NF-kappa B Transcription Factor and Human Immunodeficiency Virus Type 1 (HIV-1) Activation by Methylene Blue Photosensitization

Reactive oxygen species like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and then in the activation and replication of HIV-1 in human cells. Because singlet oxygen (1O2) is another very important reactive oxygen species whose action in transcription factor activation is totally undetermined, we started to investigate its role in both NF-kappa B and HIV-1 activation. For provoking unbalanced redox conditions, 1O2 was generated by photosensitization using methylene blue as photosensitizer. Lymphocytes or monocytes (ACH-2 or U1 respectively) latently infected with HIV-1 were treated by photosensitization mediated by methylene blue and the production of reactive oxygen species was monitored through their cytotoxic effect in infected cells. The generation of 1O2 by methylene blue turns out to be very efficient in inducing NF-kappa B as a heterodimer composed of the p50 and p65 subunits. This induction appears specific since other transcription factors like AP-1 are only weakly activated by this treatment. In comparison with other inducing treatments such as phorbol esters or tumor necrosis factor alpha (TNF-alpha), the methylene-blue-mediated activation of NF-kappa B is slow, becoming optimal 180 min after treatment. These kinetic data were obtained by following, on the same samples, both the emergence of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in the cytoplasmic extracts. Conjugated with the induction of this transcription factor, HIV-1 reactivation from these latently infected cells was also observed by the measurement of reverse transcriptase activity in the cell supernatants. These data allow us to postulate that 1O2 is a biologically important reactive oxygen species which could play a role in the establishment of oxidative stress conditions leading to HIV-1 activation via the presence of NF-kappa B in the nucleus of infected cells.

NF-kappa B Transcription Factor and Human Immunodeficiency Virus Type 1 (HIV-1) Activation by Methylene Blue Photosensitization

Reactive oxygen species like hydrogen peroxide (H2O2) have been shown to serve as messengers in the induction of NF-kappa B and then in the activation and replication of HIV-1 in human cells. Because singlet oxygen (1O2) is another very important reactive oxygen species whose action in transcription factor activation is totally undetermined, we started to investigate its role in both NF-kappa B and HIV-1 activation. For provoking unbalanced redox conditions, 1O2 was generated by photosensitization using methylene blue as photosensitizer. Lymphocytes or monocytes (ACH-2 or U1 respectively) latently infected with HIV-1 were treated by photosensitization mediated by methylene blue and the production of reactive oxygen species was monitored through their cytotoxic effect in infected cells. The generation of 1O2 by methylene blue turns out to be very efficient in inducing NF-kappa B as a heterodimer composed of the p50 and p65 subunits. This induction appears specific since other transcription factors like AP-1 are only weakly activated by this treatment. In comparison with other inducing treatments such as phorbol esters or tumor necrosis factor alpha (TNF-alpha), the methylene-blue-mediated activation of NF-kappa B is slow, becoming optimal 180 min after treatment. These kinetic data were obtained by following, on the same samples, both the emergence of NF-kappa B in the nucleus and the disappearance of I kappa B-alpha in the cytoplasmic extracts. Conjugated with the induction of this transcription factor, HIV-1 reactivation from these latently infected cells was also observed by the measurement of reverse transcriptase activity in the cell supernatants. These data allow us to postulate that 1O2 is a biologically important reactive oxygen species which could play a role in the establishment of oxidative stress conditions leading to HIV-1 activation via the presence of NF-kappa B in the nucleus of infected cells.

Inhibition of 3′Azido-3′Deoxythymidine-Resistant HIV-1 Infection by Dehydroepiandrosterone in Vitro

Human immunodeficiency virus type 1 (HIV-1) isolated from patients with acquired immunodeficiency syndrome (AIDS) shows resistance to 3′azido-3′deoxythymidine (AZT) after one or two years of treatment. AZT also has significant toxic side effects, further limiting its use in the therapy of HIV-l-infected individuals. Dehydroepiandrosterone (DHEA) has been shown to have a broad spectrum of biological functions, to be bioavailable orally and to be relatively nontoxic. Epidemiological studies provide evidence that reduced serum levels of DHEA are related to the progression of AIDS in HIV-1 infection. DHEA has also been shown to inhibit HIV-1 replication in vitro and block HIV-1 reactivation from chronically infected cell lines. However, there have been no reports on the ability of DHEA to inhibit the replication of AZT-resistant strains of HIV-1. We investigated whether DHEA treatment could inhibit replication of AZT-resistant strains of HIV-1. Addition of DHEA to MT-2 cell cultures infected with either AZT-sensitive or AZT-resistant isolates of HIV-1 resulted in dose-dependent inhibition of HTV-1-induced cytopathic effect and suppression of HIV-1 replication as measured by accumulation of reverse transcriptase activity. At a concentration as low as 50 μM, DHEA reduced AZT-resistant HIV-1 replication over 50 percent as measured by cytopathic effect and accumulation of reverse transcriptase activity. This study provides evidence that DHEA can inhibit the replication of AZT-resistant as well as wild-type HIV-1. Since the main targets for DHEA are metabolic and cellular signaling pathways leading to HIV-1 replication-activation, DHEA should be effective against multidrug-resistant strains of HIV-1. Combined with recently discovered immunoregulatory properites, the finding that DHEA is able to inhibit replication of both wild-type and AZT-resistant HIV-1 suggests that in vivo DHEA may have a much broader spectrum of action than originally anticipated.

Inhibition of human immunodeficiency virus reactivation from latency by a tat transdominant negative mutant

A BK virus (BKV) expression vector, specific for human cells, was engineered to express antisense human immunodeficiency virus type 1 (HIV-1) tat cDNA (tat-AS) or a tat mutant in cysteine 22 (tat22). Cysteine residues in the cysteine-rich domain of tat are necessary for tat transactivation of the HIV-1 long terminal repeat (LTR). Both the AS tat and the tat mutant significantly inhibited transactivation by tat when assayed in cells cotransfected with an expression vector where the reporter gene for chloramphenicol acetyl transferase was driven by the HIV-1 LTR. Infection of Jurkat cell clones stably expressing tat22 (Jurkat/tat22) or tat-AS (Jurkat/tat-AS) with HIV-1 did not show differences in virus titer in comparison to HIV-1-infected control cells. However, in two Jurkat/tat22 cell clones, entrance of HIV-1 into latency was accelerated significantly and reactivation of HIV-1 from latency induced by tumor necrosis factor-alpha (TNF-alpha) or tat was blocked. These results suggest that, in a combined and integrated approach to the treatment of acquired immunodeficiency syndrome (AIDS), anti-tat genetic therapy could be successfully applied to maintain virus in latency, thereby extending the duration of the asymptomatic phase preceding full-blown AIDS.

Inhibition of HIV-1 Latency Reactivation by Dehydroepiandrosterone (DHEA) and an Analog of DHEA

The initial infection with human immunodeficiency virus type 1 (HIV-1) in most individuals usually results in the establishment of a latent or chronic infection before eventual progression toward acquired immunodeficiency syndrome. HIV-1 can also establish a latent or persistent infection in some T cell lines that show minimal constitutive virus expression. However, activation of the T cell lines leading to enhanced HIV-1 replication can be induced by antigens, mitogens, and cytokines (tumor necrosis factor alpha [TNF-alpha], interleukin 1, and interleukin-2). Various gene products from other viruses (HTLV-1, HSV, EBV, CMV, HBV, and HHV-6) can also enhance HIV-1 long terminal repeat (LTR)-driven reporter gene activity. On the basis of these observations, it has been proposed that reactivation of latent HIV-1 harbored in chronically infected T lymphocytes, monocytes, or macrophages plays an important role in the pathogenesis of AIDS. So far, there are no drugs or therapy available that can provide protection against HIV-1 latency reactivation. ACH-2, derived from a human T cell line (CEM), is chronically infected with HIV-1, with low levels of constitutive virus expression. ACH-2 can be converted to productive infection by stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), mitogen or cytokines (TNF-alpha), or infection with HSV. Therefore the ACH-2 cell line is a good candidate for studying the effects of drugs on HIV-1 activation. Previously, we have reported that DHEA and synthetic analogs of DHEA can be modest inhibitors of HIV-1 IIIB replication in phytohemagglutinin-stimulated peripheral blood lymphocyte cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

HIV-1 promoter activation following an oxidative stress mediated by singlet oxygen

Various biological processes, such as photosensitization or inflammatory reactions, can generate singlet oxygen ( 1O 2) as one of the major oxidative species. Because this oxidant can be generated either extracellularly or intracellularly, it can cause severe damage to various biological macromolecules, even to those deeply embedded inside the cells such as DNA. Sublethal biological modifications induced by different DNA-damaging agents can promote various cellular responses initiated by the activation of various cellular genes and certain heterologous viruses. Since 1O 2 fulfils essential prerequisites for a genotoxic substance, we have examined the effects of an oxidative stress, mediated by this species, on cells harbouring a heterologous promoter-leader sequence derived from the human immunodeficiency virus type 1 (HIV-1). Our results demonstrate that HIV-1 long terminal repeat (LTR), integrated into the cellular DNA of epithelial cells, can be transactivated following an oxidative stress mediated by 1O 2. In addition, using HIV-1 latently infected promonocytes or lymphocytes, it can be shown that virus reactivation can be induced through a sublethal dose of 1O 2 generated intracellularly. An extracellular generation of 1O 2 can promote a substantial lethal effect without HIV-1 reactivation. These data may be relevant to the understanding of the events converting a latent infection into a productive one and to the appearance of the acquired immune deficiency syndrome.

Oscillation of the human immunodeficiency virus surface receptor is regulated by the state of viral activation in a CD4+ cell model of chronic infection

We have developed a unique physiologic model of chronic human immunodeficiency virus type 1 (HIV-1) infection, OM-10.1, clonally derived from infected HL-60 promyelocytes and harboring a single integrated provirus. Unlike other models of chronic infection, OM-10.1 cultures remain CD4+ under normal culture conditions, during which less than 10% of the cells constitutively express HIV-1 proteins. However, when treated with tumor necrosis factor alpha (TNF-alpha), OM-10.1 cultures dramatically increased (greater than 35-fold) HIV-1 expression and rapidly down-modulated surface CD4, as greater than 95% of the cells became HIV-1+. The complete loss of surface CD4 following viral activation was neither associated with apparent cytopathicity nor due to a decline of available CD4 mRNA. There was, however, a temporal association between CD4 down-modulation and the accumulation of intracellular HIV-1 gp 160/120; in addition, intracellular CD4-gp 160 complexes were identifiable in OM-10.1 cell lysates at time points following TNF-alpha induction after surface CD4 was no longer detectable. Surface CD4 expression by OM-10.1 cells returned once viral activation ceased and could be repeatedly oscillated upon HIV-1 reactivation. Furthermore, inhibition of protein kinase activity following maximal TNF-alpha stimulation of OM-10.1 cells quickly returned activated HIV-1 to a state of latency, as evidenced by an accelerated return of surface CD4. These results with the new OM-10.1 cell line demonstrate that CD4 surface expression can be maintained during chronic infection and is critically dependent on the state of viral activation, that CD4-gp 160 intracellular complexing is involved in CD4 down-modulation, and that protein kinase pathways not only function in the primary induction of latent HIV-1 but also are required for maintaining the state of viral activation.