Rapid Clearance of Simian Immunodeficiency Virus Particles from Plasma of Rhesus Macaques

Perturbation of the equilibrium between human immunodeficiency virus type 1 (HIV-1) and the infected host by administering antiretroviral agents has revealed the rapid turnover of both viral particles and productively infected cells. In this study, we used the infusion of simian immunodeficiency virus (SIV) particles into rhesus macaques to obtain a more accurate estimate of viral clearance in vivo. Consistently, exogenously infused virions were cleared from plasma with an extremely short half-life, on the order of minutes (a mean of 3.3 min). This new estimate is ~100-fold lower than the upper bound of 6 h previously reported for HIV-1 in infected humans. In select animals, multiple tissues were collected at the completion of each experiment to track the potential sites of virion clearance. Detectable levels of SIV RNA were found in lymph nodes, spleen, lungs, and liver, but not in other tissues examined. However, only ~1 to 10% or less of the infused virions were accounted for by the thorough tissue sampling, indicating that the vast majority of the infused particles must have been degraded over a short period of time. Should the rapid clearance of virions described here be applicable to infected patients, then HIV-1 production and thus the number of productively infected CD4⁺ T lymphocytes or the viral burst size must be proportionally higher than previous minimal estimates.     

Autophagy plays an important role in the containment of HIV-1 in nonprogressor-infected patients

Recent in vitro studies have suggested that autophagy may play a role in both HIV-1 replication and disease progression. In this study we investigated whether autophagy protects the small proportion of HIV-1 infected individuals who remain clinically stable for years in the absence of antiretroviral therapy, these named long-term nonprogressors (LTNP) and elite controllers (EC). We found that peripheral blood mononuclear cells (PBMC) of the HIV-1 controllers present a significantly higher amount of autophagic vesicles associated with an increased expression of autophagic markers with respect to normal progressors. Of note, ex vivo treatment of PBMC from the HIV-1 controllers with the MTOR inhibitor rapamycin results in a more efficient autophagic response, leading to a reduced viral production. These data lead us to propose that autophagy contributes to limiting viral pathogenesis in HIV-1 controllers by targeting viral components for degradation.     

Statin-induced inhibition of HIV-1 release from latently infected U1 cells reveals a critical role for protein prenylation in HIV-1 replication.

Latent infection of human immunodeficiency virus type 1 (HIV-1) represents a major hurdle in the treatment of acquired immunodeficiency syndrome (AIDS) patients. Statins were recently reported to suppress acute HIV-1 infection and reduce infectious virion production, but the precise mechanism of inhibition has remained elusive. Here we demonstrate that lypophilic statins suppress HIV-1 virion release from tumor necrosis factor alpha-stimulated latently infected U1 cells through inhibition of protein geranylgeranylation, but not by cholesterol depletion. Indeed, this suppression was reversed by the addition of geranylgeranylpyrophosphate, and a geranylgeranyltransferase-1 inhibitor reduced HIV-1 production. Notably, silencing of the endogenous Rab11a GTPase expression in U1 cells by RNA interference destabilized Gag and reduced virion production both in vitro and in NOD/SCID/gammac null mice. Our findings thus suggest that small GTPase proteins play an important role in HIV-1 replication, and therefore could be attractive molecular targets for anti-HIV-1 therapy.     

Gag-Pol Processing during HIV-1 Virion Maturation: A Systems Biology Approach

Proteolytic processing of Gag and Gag-Pol polyproteins by the viral protease (PR) is crucial for the production of infectious HIV-1, and inhibitors of the viral PR are an integral part of current antiretroviral therapy. The process has several layers of complexity (multiple cleavage sites and substrates; multiple enzyme forms; PR auto-processing), which calls for a systems level approach to identify key vulnerabilities and optimal treatment strategies. Here we present the first full reaction kinetics model of proteolytic processing by HIV-1 PR, taking into account all canonical cleavage sites within Gag and Gag-Pol, intermediate products and enzyme forms, enzyme dimerization, the initial auto-cleavage of full-length Gag-Pol as well as self-cleavage of PR. The model allows us to identify the rate limiting step of virion maturation and the parameters with the strongest effect on maturation kinetics. Using the modelling framework, we predict interactions and compensatory potential between individual cleavage rates and drugs, characterize the time course of the process, explain the steep dose response curves associated with PR inhibitors and gain new insights into drug action. While the results of the model are subject to limitations arising from the simplifying assumptions used and from the uncertainties in the parameter estimates, the developed framework provides an extendable open-access platform to incorporate new data and hypotheses in the future.     

Theoretical design of a gene therapy to prevent AIDS but not human immunodeficiency virus type 1 infection

Recent reports confirm that, due to the presence of long-lived, latently infected cell populations, eradication of human immunodeficiency virus type 1 (HIV-1) from infected patients by using antiretroviral drugs will be exceedingly difficult. An alternative to virus eradication may be to use gene therapy to induce a pseudo-latent state in virus-producing cells, thus transforming HIV-1 into a lifelong, but manageable, virus. Conditionally replicating HIV-1 (crHIV-1) gene therapy vectors provide an avenue for subduing HIV-1 expression in infected cells (by creating a parasite, crHIV-1, of the parasite HIV-1), potentially reducing the HIV-1 set point and delaying AIDS onset. Development of crHIV-1 vectors has proceeded in vitro, but the requirements for a crHIV-1 vector to proliferate and persist in vivo have not been explored. We expand a widely accepted mathematical model of HIV-1 in vivo dynamics to include a crHIV-1 gene therapy virus and derive a simple criterion for designing crHIV-1 viruses that will persist in vivo. The model introduces only two new parameters-HIV-1 inhibition and crHIV-1 production-and both can be experimentally engineered and controlled. Analysis demonstrates that crHIV-1 gene therapy can indefinitely reduce HIV-1 set point to levels comparable to those achieved with highly active antiretroviral therapy, provided crHIV-1 production is more efficient than HIV-1. Paradoxically, highly efficient therapeutic inhibition of HIV-1 was found to be disadvantageous. Thus, the field may benefit by shifting the search for more potent antiviral genes toward engineering optimized therapy viruses that package ultra-efficiently while downregulating viral production moderately.     

HIV病毒潜伏库的形成机制和对策挑战

长期以来,病毒潜伏库(latent viral reservoir,LVR)的存在严重阻碍了AIDS的有效治疗,LVR无法被人体免疫系统识别,高效抗逆转录病毒疗法(highly active antiretroviral therapy, HAART)对其无效,一旦中断抗病毒治疗,患者会出现快速耐药和病毒血症反弹.截至...     

Phenotypic patterns of HIV-1 clonal populations during highly active antiretroviral therapy (HAART).

Several studies have demonstrated that during HIV-1 infection many different viral clones may co-exist in the same individual. These clones may differ regarding their biological phenotype and may influence both the natural history of infection and the clinical response to antiretroviral therapy. The aim of the present study was to investigate the influences of combination therapies including protease inhibitors (HAART) on the phenotypical pattern of HIV-1 biological clones in peripheral blood mononuclear cells. Viral isolation and biological characterisation of bulk isolates and clonal viral isolates were performed on two AIDS patients, before and after three months of antiretroviral therapy. A decrease of viral load in plasma specimens in association with a change of clonal composition during antiretroviral therapy was observed in both patients during treatment. Before therapy both of the patients had a syncytium inducing (SI) bulk isolate and the majority of the biological clones were characterised as SI. After treatment, the bulk isolates were still SI in both cases, but the majority of biological clones were characterised as non-syncytium inducing (NSI). These results suggest that HIV clonal composition and relative phenotypic pattern undergo different changes not only during the natural course of HIV infection but also while patients are on antiretroviral combination therapy.     

Circulating monocytes are not a major reservoir of HIV-1 in elite suppressors

Circulating HIV-1-infected monocytes have been identified in patients on highly active antiretroviral therapy and may represent an important barrier to viral eradication. The nature of these cells in HIV-1-infected patients who maintain undetectable viral loads and preserved CD4(+) T cell counts without antiretroviral therapy (known as elite controllers or elite suppressors [ES]) is unknown. We describe here infrequent recovery of proviral HIV-1 DNA from circulating monocytes relative to CD4(+) T cells in ES, despite permissiveness of these cells to HIV-1 viral entry ex vivo. Thus, monocytes do not appear to be a major reservoir of HIV-1 in ES.     

Reconstitution of Human Thymic Implants Is Limited by Human Immunodeficiency Virus Breakthrough during Antiretroviral Therapy

Human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu thymic implants depleted of CD4(+) cells can support renewed thymopoiesis derived from both endogenous and exogenous T-cell progenitors after combination antiretroviral therapy. However, successful production of new thymocytes occurs transiently. Possible explanations for the temporary nature of this thymic reconstitution include cessation of the thymic stromal support function, exhaustion of T-cell progenitors, and viral resurgence. Distinguishing between these processes is important for the development of therapeutic strategies aimed at reconstituting the CD4(+) T-cell compartment in HIV-1 infection. Using an HIV-1 strain engineered to express the murine HSA heat-stable antigen surface marker, we explored the relationship between HIV-1 expression and CD4(+) cell resurgence kinetics in HIV-1-depleted SCID-hu implants following drug therapy. Antiviral therapy significantly suppressed HIV-1 expression in double-positive (DP) CD4/CD8 thymocytes, and the eventual secondary decline of DP thymocytes following therapy was associated with renewed viral expression in this cell subset. Thymocytes derived from exogenous T-cell progenitors induced to differentiate in HIV-1-depleted, drug-treated thymic implants also became infected. These results indicate that in this model, suppression of viral replication occurs transiently and that, in spite of drug therapy, virus resurgence contributes to the transient nature of the renewed thymic function.     

Modeling within-host HIV-1 dynamics and the evolution of drug resistance: trade-offs between viral enzyme function and drug susceptibility

There are many biological steps between viral infection of CD4(+) T cells and the production of HIV-1 virions. Here we incorporate an eclipse phase, representing the stage in which infected T cells have not started to produce new virus, into a simple HIV-1 model. Model calculations suggest that the quicker infected T cells progress from the eclipse stage to the productively infected stage, the more likely that a viral strain will persist. Long-term treatment effectiveness of antiretroviral drugs is often hindered by the frequent emergence of drug resistant virus during therapy. We link drug resistance to both the rate of progression of the eclipse phase and the rate of viral production of the resistant strain, and explore how the resistant strain could evolve to maximize its within-host viral fitness. We obtained the optimal progression rate and the optimal viral production rate, which maximize the fitness of a drug resistant strain in the presence of drugs. We show that the window of opportunity for invasion of drug resistant strains is widened for a higher level of drug efficacy provided that the treatment is not potent enough to eradicate both the sensitive and resistant virus.     

Organization of Cellular Receptors into a Nanoscale Junction during HIV-1 Adhesion

The fusion of the human immunodeficiency virus type 1 (HIV-1) with its host cell is the target for new antiretroviral therapies. Viral particles interact with the flexible plasma membrane via viral surface protein gp120 which binds its primary cellular receptor CD4 and subsequently the coreceptor CCR5. However, whether and how these receptors become organized at the adhesive junction between cell and virion are unknown. Here, stochastic modeling predicts that, regarding binding to gp120, cellular receptors CD4 and CCR5 form an organized, ring-like, nanoscale structure beneath the virion, which locally deforms the plasma membrane. This organized adhesive junction between cell and virion, which we name the viral junction, is reminiscent of the well-characterized immunological synapse, albeit at much smaller length scales. The formation of an organized viral junction under multiple physiopathologically relevant conditions may represent a novel intermediate step in productive infection.     

Current Estimates for HIV-1 Production Imply Rapid Viral Clearance in Lymphoid Tissues

It has recently been estimated that a single HIV-1 infected cell produces between and more than viral particles over its life span. Since body-wide estimates of the ratio of free virus to productively infected cells are smaller than and much smaller than , individual virions must be cleared rapidly. This seems difficult to reconcile with the fact that most of the total body virus is trapped on follicular dendritic cells where it can survive for many months. It has also been difficult to reconcile the vast difference in the rates at which the virus is cleared from the blood in rhesus macaques and in chronically infected patients. Here we attempt to reconcile these seemingly contradictory observations by considering the virion clearance rate in various organs and the virion exchange rates between them. The main results are that the per capita clearance rate of free virus in lymphoid tissue should be fast, the virion exchange rate between lymphoid tissue and the blood should be slow, and the comparatively slow previous estimates for the virion clearance rate from the blood correspond to the rate of virion efflux from the blood to other organs where the virus is ultimately cleared.     

Criticality of timing for anti-HIV therapy initiation

The time of initiation of antiretroviral therapy in HIV-1 infected patients has a determinant effect on the viral dynamics. The question is, how far can the therapy be delayed? Is sooner always better? We resort to clinical data and to microsimulations to forecast the dynamics of the viral load at therapy interruption after prolonged antiretroviral treatment. A computational model previously evaluated, produces results that are statistically adherent to clinical data. In addition, it allows a finer grain analysis of the impact of the therapy initiation point to the disease course. We find a swift increase of the viral density as a function of the time of initiation of the therapy measured when the therapy is stopped. In particular there is a critical time delay with respect to the infection instant beyond which the therapy does not affect the viral rebound. Initiation of the treatment is beneficial because it can down-regulate the immune activation, hence limiting viral replication and spread.     

A novel assay allows genotyping of the latent reservoir for human immunodeficiency virus type 1 in the resting CD4+ T cells of viremic patients

A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.