Miguel Muñoz

Individual Contributions of Mutant Protease and Reverse Transcriptase to Viral Infectivity, Replication, and Protein Maturation of Antiretroviral Drug-Resistant Human Immunodeficiency Virus Type 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) variants resistant to protease (PR) and reverse transcriptase (RT) inhibitors may display impaired infectivity and replication capacity. The individual contributions of mutated HIV-1 PR and RT to infectivity, replication, RT activity, and protein maturation (herein referred to as “fitness”) in recombinant viruses were investigated by separately cloning PR, RT, and PR-RT cassettes from drug-resistant mutant viral isolates into the wild-type NL4-3 background. Both mutant PR and RT contributed to measurable deficits in fitness of viral constructs. In peripheral blood mononuclear cells, replication rates (means ± standard deviations) of RT recombinants were 72.5% ± 27.3% and replication rates of PR recombinants were 60.5% ± 33.6% of the rates of NL4-3. PR mutant deficits were enhanced in CEM T cells, with relative replication rates of PR recombinants decreasing to 15.8% ± 23.5% of NL4-3 replication rates. Cloning of the cognate RT improved fitness of some PR mutant clones. For a multidrug-resistant virus transmitted through sexual contact, RT constructs displayed a marked infectivity and replication deficit and diminished packaging of Pol proteins (RT content in virions diminished by 56.3% ± 10.7%, and integrase content diminished by 23.3% ± 18.4%), a novel mechanism for a decreased-fitness phenotype. Despite the identified impairment of recombinant clones, fitness of two of the three drug-resistant isolates was comparable to that of wild-type, susceptible viruses, suggestive of extensive compensation by genomic regions away from PR and RT. Only limited reversion of mutated positions to wild-type amino acids was observed for the native isolates over 100 viral replication cycles in the absence of drug selective pressure. These data underscore the complex relationship between PR and RT adaptive changes and viral evolution in antiretroviral drug-resistant HIV-1.

Resistance to Nucleoside Analog Reverse Transcriptase Inhibitors Mediated by Human Immunodeficiency Virus Type 1 p6 Protein

ABSTRACT Resistance of human immunodeficiency virus type 1 (HIV-1) to antiretroviral agents results from target gene mutation within thepol gene, which encodes the viral protease, reverse transcriptase (RT), and integrase. We speculated that mutations in genes other that the drug target could lead to drug resistance. For this purpose, the p1-p6 gag -p6 pol region of HIV-1, placed immediately upstream ofpol, was analyzed. This region has the potential to alter Pol through frameshift regulation (p1), through improved packaging of viral enzymes (p6Gag), or by changes in activation of the viral protease (p6Pol). Duplication of the proline-rich p6Gag PTAP motif, necessary for late viral cycle activities, was identified in plasma virus from 47 of 222 (21.2%) patients treated with nucleoside analog RT inhibitor (NRTI) antiretroviral therapy but was identified very rarely from drug-naı̈ve individuals. Molecular clones carrying a 3-amino-acid duplication, APPAPP (transframe duplication SPTSPT in p6Pol), displayed a delay in protein maturation; however, they packaged a 34% excess of RT and exhibited a marked competitive growth advantage in the presence of NRTIs. This phenotype is reminiscent of the inoculum effect described in bacteriology, where a larger input, or a greater infectivity of an organism with a wild-type antimicrobial target, leads to escape from drug pressure and a higher MIC in vitro. Though the mechanism by which the PTAP region participates in viral maturation is not known, duplication of this proline-rich motif could improve assembly and packaging at membrane locations, resulting in the observed phenotype of increased infectivity and drug resistance.

Mycobacterium tuberculosis subverts innate immunity to evade specific effectors

The macrophage is the niche of the intracellular pathogen Mycobacterium tuberculosis. Induction of macrophage apoptosis by CD4+ or CD8+ T cells is accompanied by reduced bacterial counts, potentially defining a host defense mechanism. We have already established that M. tuberculosis-infected primary human macrophages have a reduced susceptibility to Fas ligand (FasL)-induced apoptosis. To study the mechanisms by which M. tuberculosis prevents apoptotic signaling, we have generated a cell culture system based on PMA- and IFN-γ-differentiated THP-1 cells recapitulating the properties of primary macrophages. In these cells, nucleotide-binding oligomerization domain 2 or TLR2 agonists and mycobacterial infection protected macrophages from apoptosis and resulted in NF-κB nuclear translocation associated with up-regulation of the antiapoptotic cellular FLIP. Transduction of a receptor-interacting protein-2 dominant-negative construct showed that nucleotide-binding oligomerization domain 2 is not involved in protection in the mycobacterial infection system. In contrast, both a dominant-negative construct of the MyD88 adaptor and an NF-κB inhibitor abrogated the protection against FasL-mediated apoptosis, showing the implication of TLR2-mediated activation of NF-κB in apoptosis protection in infected macrophages. The apoptosis resistance of infected macrophages might be considered as an immune escape mechanism, whereby M. tuberculosis subverts innate immunity signaling to protect its host cell against FasL+-specific cytotoxic lymphocytes.

24 Hours in the Life of HIV-1 in a T Cell Line

HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.

Immunological and virological responses in HIV-1-infected adults at early stage of established infection treated with highly active antiretroviral therapy.

ObjectiveTo evaluate the immunological and virological responses to highly active antiretroviral therapy (HAART) in blood and lymphoid compartments of HIV-1-infected patients at an early stage of infection. DesignAn open-label, observational, non-randomized, prospective trial of outpatients attending the Centre of Clinical Investigation in Infectious Diseases, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Switzerland. SubjectsForty-one antiretroviral-naive HIV-1-infected adults with 400 CD4 T cells/μl or greater and 5000 plasma HIV-1-RNA copies/ml or greater were enrolled, and 32 finished the study. Forty-nine HIV-negative individuals were included as controls. All subjects gave written informed consent. InterventionsAll patients received abacavir 300 mg by mouth every 12 h and amprenavir 1200 mg by mouth every 12 h for 72 weeks. Mainoutcome measuresThe extent of immune reconstitution in blood and lymph nodes after 72 weeks of HAART was evaluated, and compared with immunological measures of 49 HIV-negative subjects. ResultsVirus replication was effectively suppressed (−3.5 log10 at week 72). Substantial increments of CD4 T cell count in blood and percentage in lymph nodes were observed over time, and these measures were comparable to HIV-negative subjects by week 24 in blood and by week 48 in lymph nodes. The increase was equally distributed between naive and memory CD4 T cells. Recovery of HIV-specific CD4 responses occurred in 40% of patients. ConclusionThe initiation of HAART at an early stage of established HIV infection induces systemic quantitative normalization of CD4 T cells, a partial recovery of HIV-specific CD4 cell responses, and effective and durable suppression of virus replication.

In vitro whole-genome analysis identifies a susceptibility locus for HIV-1

Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.

Analysis of natural variants of the human immunodeficiency virus type 1 gag-pol frameshift stem-loop structure.

ABSTRACT Human immunodeficiency virus type 1 uses ribosomal frameshifting for translation of the Gag-Pol polyprotein. Frameshift activities are thought to be tightly regulated. Analysis of gag p1 sequences from 270 plasma virions identified in 64% of the samples the occurrence of polymorphism that could lead to changes in thermodynamic stability of the stem-loop. Expression in Saccharomyces cerevisiae of p1-β-galactosidase fusion proteins from 10 representative natural stem-loop variants and three laboratory mutant constructs (predicted the thermodynamic stability [ΔG°] ranging from −23.0 to −4.3 kcal/mol) identified a reduction in frameshift activity of 13 to 67% compared with constructs with the wild-type stem-loop (ΔG°, −23.5 kcal/mol). Viruses carrying stem-loops associated with greater than 60% reductions in frameshift activity presented profound defects in viral replication. In contrast, viruses with stem-loop structures associated with 16 to 42% reductions in frameshift efficiency displayed no significant viral replication deficit.

Analysis of HIV-1 expression level and sense of transcription by high-throughput sequencing of the infected cell

ABSTRACT Next-generation sequencing offers an unprecedented opportunity to jointly analyze cellular and viral transcriptional activity without prerequisite knowledge of the nature of the transcripts. SupT1 cells were infected with a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped HIV vector. At 24 h postinfection, both cellular and viral transcriptomes were analyzed by serial analysis of gene expression followed by high-throughput sequencing (SAGE-Seq). Read mapping resulted in 33 to 44 million tags aligning with the human transcriptome and 0.23 to 0.25 million tags aligning with the genome of the HIV-1 vector. Thus, at peak infection, 1 transcript in 143 is of viral origin (0.7%), including a small component of antisense viral transcription. Of the detected cellular transcripts, 826 (2.3%) were differentially expressed between mock- and HIV-infected samples. The approach also assessed whether HIV-1 infection modulates the expression of repetitive elements or endogenous retroviruses. We observed very active transcription of these elements, with 1 transcript in 237 being of such origin, corresponding on average to 123,123 reads in mock-infected samples (0.40%) and 129,149 reads in HIV-1-infected samples (0.45%) mapping to the genomic Repbase repository. This analysis highlights key details in the generation and interpretation of high-throughput data in the setting of HIV-1 cellular infection.

Entry and Transcription as Key Determinants of Differences in CD4 T-Cell Permissiveness to Human Immunodeficiency Virus Type 1 Infection

ABSTRACT Isolated primary human cells from different donors vary in their permissiveness—the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4+ CD45RO+ CD57− T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

Dynamics of HIV Latency and Reactivation in a Primary CD4+T Cell Model

HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.