José A. Esté

HIV entry inhibitors

The need for new classes of antiretroviral drugs has become apparent because of increasing concern about the long-term toxic effects of existing drugs, the need to combat HIV-1 variants that are resistant to treatment, and the frequency of treatment change in drug-experienced patients. Currently, most regimens are combinations of inhibitors of two viral enzymes--reverse transcriptase and protease. Nevertheless, several steps in the HIV replication cycle are potential targets for intervention. These steps can be divided into entry steps, in which viral envelope glycoproteins and their receptors are involved, and postentry steps, involving viral accessory gene products and the cellular proteins with which they interact. New treatment options target viral entry into the cell. These treatments include the HIV fusion inhibitor enfuvirtide, and new HIV coreceptor antagonists in advanced stages of clinical development or in different stages of preclinical development. Here, we review the development of new HIV entry inhibitors, their performance in clinical trials, and their possible role in anti-HIV therapy.

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.

Bicyclams, a class of potent anti-HIV agents, are targeted at the HIV coreceptor fusin/CXCR-4.

Bicyclams are a novel class of antiviral compounds which are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. The prototype compound, AMD3100, has an IC50 of 1-10 ng/ml, which is a least 100,000 fold lower than the cytotoxic concentration. AMD3100 does not inhibit virus binding to the CD4 receptor and based on time-of-addition experiments, has been assumed to interact with the HIV fusion-uncoating process. Resistance of HIV-1 strains to AMD3100 is associated with the accumulation of several mutations in the viral envelope glycoprotein gp120. Here, we demonstrate that AMD3100 interacts with fusin (CXCR-4), the coreceptor used by T-tropic viruses to infect the target cells. The replication of NL4-3 wild type virus and NL4-3 dextran sulfate-resistant virus was inhibited by the CXC-chemokine, stromal cell-derived factor 1 (SDF-1), the natural ligand for CXCR-4. In contrast, the replication of the HIV-1 NL4-3 AMD3100-resistant virus was no longer inhibited by SDF-1. The bicyclams are the first low-molecular-weight anti-HIV agents shown to interact with the coreceptor for T-tropic viruses.

Current status and challenges of antiretroviral research and therapy.

Twenty-five years after the discovery of the therapeutic activity of azidothymidine (AZT), the first antiretroviral drug used in the clinic, infection with the human immunodeficiency virus (HIV) has become, at least in the industrialized world, a manageable chronic disease with a significant improvement in life expectancy and quality. Nevertheless, the number of new infections worldwide continues to rise, particularly in women, and effective drug treatments have not yet reached the vast majority of infected individuals in resource-limited countries. The current status of antiretroviral therapy is therefore encouraging, but significant challenges remain. Although highly active antiretroviral therapy (HAART) provides durable control of virus replication in many patients, it is not devoid of unwanted secondary effects, some of which are now surfacing in aging populations under long-term treatment. The emergence of multidrug resistance and transmission of drug-resistant HIV strains limit the clinical efficacy of current therapy. Further simplification of treatment and identification of more effective drug combinations are needed to improve patient adherence, the most significant cause of treatment failure. Finding new drugs and novel drug targets may lead to redefining the goals of antiretroviral therapy, with an attempt to achieve the ultimate objective: the eradication of infection. Preclinical and clinical biomedical research, rational drug design and a close collaboration with regulatory agencies to set standards for the transition of new treatment concepts into the clinic will be the cornerstones of future progress. This special issue of Antiviral Research [85(1), 2010] highlights the principal milestones of antiretroviral research over 25 years of drug discovery and development and offers a comprehensive analysis by leading experts of the efforts being made to meet the challenges of effective control of HIV infection. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010.

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV‐1 envelope

Syncytia arising from the fusion of cells expressing the HIV‐1‐encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR‐mediated phosphorylation of p53 on Ser15 (p53S15), p53‐dependent upregulation of Bax and activation of the mitochondrial death pathway. p53S15 phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase‐like dismantling of the nuclear envelope. Inhibition of cyclin‐dependent kinase‐1 (Cdk1) prevents karyogamy, mTOR activation, p53S15 phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV‐1‐infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53S15 phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV‐1 infection of primary CD4 lymphoblasts. Thus, HIV‐1 elicits a pro‐apoptotic signal transduction pathway relying on the sequential action of cyclin B–Cdk1, mTOR and p53.

Interleukin-7 in Plasma Correlates with CD4 T-Cell Depletion and May Be Associated with Emergence of Syncytium-Inducing Variants in Human Immunodeficiency Virus Type 1-Positive Individuals

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) primary infection is characterized by the use of CCR5 as a coreceptor for viral entry, which is associated with the non-syncytium-inducing (NSI) phenotype in lymphoid cells. Syncytium-inducing (SI) variants of HIV-1 appear in advanced stages of HIV-1 infection and are characterized by the use of CXCR4 as a coreceptor. The emergence of SI variants is accompanied by a rapid decrease in the number of T cells. However, it is unclear why SI variants emerge and what factors trigger the evolution of HIV from R5 to X4 variants. Interleukin-7 (IL-7), a cytokine produced by stromal cells of the thymus and bone marrow and by keratin, is known to play a key role in T-cell development. We evaluated IL-7 levels in plasma of healthy donors and HIV-positive patients and found significantly higher levels in HIV-positive patients. There was a negative correlation between circulating IL-7 levels and CD4+ T-cell count in HIV-positive patients (r = −0.621;P < 0.001), suggesting that IL-7 may be involved in HIV-induced T-cell depletion and disease progression. IL-7 levels were higher in individuals who harbored SI variants and who had progressed to having CD4 cell counts of lower than 200 cells/μl than in individuals with NSI variants at a similar stage of disease. IL-7 induced T-cell proliferation and up-regulated CXCR4 expression in peripheral blood mononuclear cells in vitro. Taken together, our results suggest a role for IL-7 in the maintenance of T-cell regeneration and depletion by HIV in infected individuals and a possible relationship between IL-7 levels and the emergence of SI variants.

Novel targets for HIV therapy.

There are currently 25 drugs belonging to 6 different inhibitor classes approved for the treatment of human immunodeficiency virus (HIV) infection. However, new anti-HIV agents are still needed to confront the emergence of drug resistance and various adverse effects associated with long-term use of antiretroviral therapy. The 21st International Conference on Antiviral Research, held in April 2008 in Montreal, Canada, therefore featured a special session focused on novel targets for HIV therapy. The session included presentations by world-renowned experts in HIV virology and covered a diverse array of potential targets for the development of new classes of HIV therapies. This review contains concise summaries of discussed topics that included Vif-APOBEC3G, LEDGF/p75, TRIM 5alpha, virus assembly and maturation, and Vpu. The described viral and host factors represent some of the most noted examples of recent scientific breakthroughs that are opening unexplored avenues to novel anti-HIV target discovery and validation, and should feed the antiretroviral drug development pipeline in the near future.

High Level of Coreceptor-independent HIV Transfer Induced by Contacts between Primary CD4 T Cells

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread, requires CD4 and coreceptor expression in target cells, and may also lead to syncytium formation and cell death. Here, we show that in addition to this classical coreceptor-mediated transmission, the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped, captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5–1.0 μm) intracellular vesicles containing 1–14 virions, without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion, we describe a novel mechanism of HIV transmission, activated by the contact of infected and uninfected primary CD4 T cells, by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir.

RNA interference of HIV replication

Double-stranded RNA-mediated interference (RNAi) induces sequence-specific post-transcriptional gene silencing and has emerged as a powerful tool to silence gene expression in multiple organisms. In mammalian cells, duplexes of 21 nucleotide RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression. Recent research demonstrates the general use of siRNAs to specifically inhibit HIV-1 replication by targeting viral or cellular genes. Importantly, RNAi opens a new avenue for gene-based therapeutics.

Cell Cycle Control and HIV-1 Susceptibility Are Linked by CDK6-Dependent CDK2 Phosphorylation of SAMHD1 in Myeloid and Lymphoid Cells

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain–containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67+) primary CD4+ T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.