Protein Kinase C: One Pathway towards the Eradication of Latent HIV-1 Reservoirs.

An effective means to eradicate latent reservoirs in HIV-1-infected individuals remains elusive. Attempts to purge these reservoirs were undertaken over a decade ago without success. The subsequent lapse in further clinical attempts since may have been justified as our knowledge of the mechanisms which underpin the latent state still evolves. Although additional novel molecular antagonists of HIV-1 latency have subsequently been reported, these candidate agents have not been tested in human trials for reservoir ablation. This review provides an overview of the protein kinase C (PKC) pathway which can be modulated by small molecular agents to induce the expression of latent HIV-1 from within infected reservoir cells. Some of these agents have been tested against select cancers with seemingly tolerable side effects. As such, modulation of the PKC pathway may yet be a viable mechanism toward HIV-1 reservoir eradication.

In vivo effects of antiviral protein kinase C modulators on zebrafish development and survival.

Clinical interventions using protein kinase C (PKC) modulators have been proposed for eradication of HIV-1-infected cellular reservoirs which persist in patients despite prolonged antiretroviral therapy. The effects of some of these agents have not been assessed in a developing vertebrate model. This study examines the developmental and toxicological effects of these compounds on zebrafish embryos and larvae. Treatment of zebrafish through the first week of development with various PKC pathway modulators did not elicit gross physical defects or elevated incidences of death at lower doses. Higher concentrations resulted in rapid death for both later-stage embryos and larvae. Each compound had a threshold dose for lethality. The defined nonlethal doses may be useful toward assessing the effects of modulating PKC activity on zebrafish development. They may further provide some guidance for the potential dosing of PKC modulators in clinical trials toward the goal of HIV-1 reservoir eradication.

High-frequency epigenetic repression and silencing of retroviruses can be antagonized by histone deacetylase inhibitors and transcriptional activators, but uniform reactivation in cell clones is restricted by additional mechanisms.

Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(-)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(-) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(-) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(-) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(-) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

HAART-persistent HIV-1 latent reservoirs: their origin, mechanisms of stability and potential strategies for eradication.

HIV-1 infection persists despite long-term administration of highly active antiretroviral therapy (HAART). The mechanism of this persistence appears to result primarily from viral infection of CD4+ T-lymphocytes that have the ability to duplicate and revert into a quiescent state. These infected resting cells are long-lived and evade immune surveillance or clearance. The inability to eradicate this class of cells, bearing the viral DNA, suggests life-long persistence of virus in HIV-1-infected individuals, even if HAART were administered for decades. This review discusses the origins and mechanisms accounting for stability of these latent HIV-1 cellular reservoirs. It further provides an overview of recent clinical trials aimed at their eradication. There have been a limited number of immune activation (IAT) trials directed at HAART-persistent, viral reservoir eradication. These trials have not resulted in purging of these highly stable viral reservoirs though results from such efforts suggest partial effects. The properties of novel compounds that might be included into IAT eradication protocols are continuing to be evaluated and their potential for inclusion into future IAT trials will be discussed.

Seminal reservoirs during an HIV type 1 eradication trial.

Despite dramatic reduction of the levels of human immunodeficiency virus type I (HIV-1) virions in blood and seminal plasma of infected patients, highly active antiretroviral therapy (HAART) does not eradicate HIV-1. Three patients, with less than 50 copies/ml of plasma viral RNA, were enrolled in this eradication protocol. Didanosine (DDI) and hydroxyurea (HU) were added to their baseline HAART and after a month of therapy, low dose OKT3, followed by a 2-week course of interleukin 2 (IL-2), was administrated. All antiretroviral therapy was then interrupted and the three patients developed viral rebound in the peripheral blood. The V3 loop region of the HIV-1 gp120 from cell-free viral RNA and proviral DNA in blood and seminal compartments was sequenced in one patient. The two major viral isolates in semen cells were macrophage- tropic (R5) and dual-tropic (R5X4), and these isolates were also present in the PBMCs. Six months after the viral rebound, we demonstrated a shift toward dual tropism in semen cell-associated HIV-1 proviral DNA, with the first appearance of a T-lymphotropic (X4) provirus solely in this compartment. The virus responsible for the blood plasma viral rebound was never found in the semen microenvironment. This study suggests viral compartmentalization of the semen microenvironment after an intensification and stimulatory HIV-1 eradication protocol, with evidence of viral evolution.

HIV type 1 cervicovaginal reservoirs in the era of HAART.

Highly active antiretroviral therapy (HAART) does not lead to viral eradication, due to HIV-1 residual disease. We investigated whether the cervicovaginal tract serves as a viral reservoir. Seven out of eight cervicovaginal fluids were positive for cell-free HIV-1, by supersensitive reverse transcriptase-polymerase chain reactions (RT-PCR), with a detection limit of 1 copy/ml. No viral outgrowth, intracellular proviral DNA, or viral RNA was detected from cervicovaginal lavage and ecto- and endocervical cells. The cervicovaginal tract of patients on HAART is likely not a major solid tissue reservoir for HIV-1. Nonetheless, the presence of even low cell-free HIV-1 RNA in cervicovaginal secretions continues to suggest the importance of practicing protected sex, even in the era of HAART.

IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART.

The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.

The perlecan heparan sulfate proteoglycan mediates cellular uptake of HIV-1 Tat through a pathway responsible for biological activity.

Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize 125I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished 125I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.

Expression of latent HAART-persistent HIV type 1 induced by novel cellular activating agents.

The novel antitumor-promoting phorbol ester, prostratin, was evaluated for its ability to induce the expression of latent, highly active antiretroviral therapy (HAART)-persistent human immunodeficiency virus type I (HIV-1) from specific subsets of patients' peripheral blood cells. This evaluation was performed relative to the use of other cellular activating agents, such as OKT3, a monoclonal antibody against the human T cell receptor, interleukin-2 (IL-2), phytohemagglutinin (PHA), p24 antigen (HIV-1-specific capsid protein), and a molecular relative of prostratin, 12-deoxyphorbol 13-phenylacetate (DPP). Prostratin performed as efficiently as the other cellular activators at inducing the expression of latent HIV-1 from cells of patients on virally suppressive HAART. Of interest was the induction of a novel species of latent virus from the cells of an individual after exposure to the HIV-1-specific capsid protein, p24, relative to virus expression induced by several other cell activators. This suggests that a variety of agents may be available for animal model studies of lentiviral latency and clinical use to broadly induce the expression of latent, HAART-persistent HIV-1 in vivo with the goal of potential HIV-1 reservoir depletion or eradication.

HIV-1-mediated apoptosis of neuronal cells: Proximal molecular mechanisms of HIV-1-induced encephalopathy.

The induction of neuronal cell death in vivo has been recognized as a prominent feature of HIV type I (HIV-1) infection leading to HIV-1-induced encephalopathy. Viral and host cell products, released from HIV-1-infected cells, have been implicated as inducers of neuronal cell apoptosis. It is unclear which is more important in this process. Neuronal cells were treated with media bearing HIV-1 virions derived from infected T cells and macrophage or the same set of media depleted of virions. T cell media bearing virus induced high levels of apoptosis, whereas that depleted of virions did not. In contrast, neurons treated with media from infected macrophages induced cell death whether virions were present or depleted by ultracentrifugation. The former initiated a repeatedly and significantly higher degree of apoptosis. These data suggest that exposure of neurons to viral products is critical for the induction of apoptosis, in addition to putative host factors released from virally infected cells. Protein-array analyses identified host cell factors up-regulated from infected macrophages, versus their uninfected counterparts, and these host cell factors may be prime candidates for contributing to neuronal apoptosis. Gene-array analyses also identified mRNAs up-regulated in human neurons after treatment with purified HIV-1 gp120 envelope protein or virus-containing media from HIV-1-infected macrophages. These analyses suggest molecular mechanisms for the induction of apoptosis relating to the exposure of viral and host cell factors and rationally designed approaches toward neuroprotection.

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.

In a subset of subjects on highly active antiretroviral therapy, human immunodeficiency virus type 1 RNA in plasma decays from 50 to <5 copies per milliliter, with a half-life of 6 months.

Three of five virally suppressed human immunodeficiency virus type I (HIV-1)-infected patients treated with highly active antiretroviral therapy and followed intensively with a supersensitive reverse transcriptase PCR assay with a lower limit of quantitation of 5 copies/ml showed statistically significant viral load decays below 50 copies/ml, with half-lives of 5 to 8 months and a mean of 6 months. This range of half-lives is consistent with the estimated half-life of the latent HIV-1 reservoir in the peripheral blood. Those patients without decay of viral load in plasma may have significant cryptic HIV-1 residual replication.

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.

Approaching eradication of highly active antiretroviral therapy-persistent human immunodeficiency virus type 1 reservoirs with immune activation therapy.

Highly active antiretroviral therapy (HAART) has dramatically altered the human immunodeficiency virus type 1 (HIV-1) pandemic in the developed world. Most patients treated with HAART will maintain clinically undetectable plasma virus loads with concomitant dramatic decreases in mortality and morbidity. Nevertheless, HAART does not eradicate HIV-1 infection on the basis of persistent low-level or cryptic viral replication and, of importance, latent provirus in resting CD4+ T lymphocytes. New approaches are now being developed for stimulation of "HAART-persistent" reservoirs. Immune activation therapy (IAT) has begun to be used in attempts to stimulate the HIV-1 latent reservoir. These studies and new approaches to activating latent virus in resting CD4+ T cells are reviewed and critically analyzed in the present report. Development of novel IAT may lead to long-term remission or viral eradication in the future.

Intensification and stimulation therapy for human immunodeficiency virus type 1 reservoirs in infected persons receiving virally suppressive highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) has led to significant changes in mortality and morbidity in the human immunodeficiency virus type 1 (HIV-1) epidemic. Nevertheless, because of molecular mechanisms of viral persistence, HAART does not eradicate HIV-1. Didanosine and hydroxyurea were added to the antiretroviral regimens of 3 HIV-1-infected men who were receiving stable HAART and who had HIV-1 RNA levels <50 copies/mL at the initiation of the study protocol, as a novel intensification to attack cryptic viral replication; low-dose OKT3 was then administered, followed by a course of interleukin-2, to stimulate latent provirus. Replication-competent virus was undetectable after treatment, and plasma viral RNA was either undetectable or <5 copies/mL. In trial periods during which no antiretroviral therapy was administered, the patients developed plasma viral rebound. This translational approach combines novel intensification and stimulation therapy to deplete residual HIV-1 reservoirs. Additional experimental approaches must be developed if HIV-1 eradication is to become possible in patients receiving virally suppressive HAART.

Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons.

Our recent studies have demonstrated that extracellular, recombinant human immunodeficiency virus type I (HIV-1) Vpr protein is highly neurotoxic in the microenvironment of differentiated mature human neurons and undifferentiated neuronal precursors. Although most of the direct neurotoxic effects of HIV-1 have been attributed previously to the envelope gene product, gp120, and the Tat regulatory protein, it was demonstrated that Vpr protein caused apoptosis comparable to that induced by gp120 protein in a dose-dependent manner in the neuronal system. Having observed the neurocytopathic effects of extracellular Vpr protein previously, the effects of virally expressed Vpr on nondividing, terminally differentiated human neurons were investigated. An HIV-1-based three-plasmid expression vector system was utilized to study the effects of intracellularly expressed Vpr. These virion preparations were then used to transduce neurons generated from the human neuronal precursor NT2 cell-line. Intracellularly expressed Vpr induced apoptosis within terminally differentiated neurons, as demonstrated by TUNEL assays. Additionally, virions lacking Vpr expression did not significantly induce apoptosis within these neurons. These results suggest that HIV-1 Vpr may also be leading directly to selective neurotoxicity through intracellular expression. Furthermore, human apoptosis gene microarray comparisons exhibited an up-regulation of Bcl-2-related mRNA, as well as other apoptosis genes involved in the mitochondrial apoptotic pathway, for the Vpr-transduced neuronal cells, when compared to Vpr-negative controls. Thus, Vpr delivered intracellularly, as well as extracellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy.