Lachlan Robert Gray

Both CD31+ and CD31- Naive CD4+ T Cells Are Persistent HIV Type 1-Infected Reservoirs in Individuals Receiving Antiretroviral Therapy

BACKGROUND Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31⁻ naive T cells to immune reconstitution and viral persistence is unknown. METHODS In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31⁻ naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31⁻ naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS After ART, both CD31(+) and CD31⁻ naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.

HIV infection of dendritic cells subverts the IFN induction pathway via IRF-1 and inhibits type 1 IFN production.

Many viruses have developed mechanisms to evade the IFN response. Here, HIV-1 was shown to induce a distinct subset of IFN-stimulated genes (ISGs) in monocyte-derived dendritic cells (DCs), without detectable type I or II IFN. These ISGs all contained an IFN regulatory factor 1 (IRF-1) binding site in their promoters, and their expression was shown to be driven by IRF-1, indicating this subset was induced directly by viral infection by IRF-1. IRF-1 and -7 protein expression was enriched in HIV p24 antigen-positive DCs. A HIV deletion mutant with the IRF-1 binding site deleted from the long terminal repeat showed reduced growth kinetics. Early and persistent induction of IRF-1 was coupled with sequential transient up-regulation of its 2 inhibitors, IRF-8, followed by IRF-2, suggesting a mechanism for IFN inhibition. HIV-1 mutants with Vpr deleted induced IFN, showing that Vpr is inhibitory. However, HIV IFN inhibition was mediated by failure of IRF-3 activation rather than by its degradation, as in T cells. In contrast, herpes simplex virus type 2 markedly induced IFNβ and a broader range of ISGs to higher levels, supporting the hypothesis that HIV-1 specifically manipulates the induction of IFN and ISGs to enhance its noncytopathic replication in DCs.

Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

BackgroundCCR5-restricted (R5) human immunodeficiency virus type 1 (HIV-1) variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env) determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA) and AIDS (A) R5 Envs, respectively.ResultsCompared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362), a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs) residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure.ConclusionEnhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

An altered and more efficient mechanism of CCR5 engagement contributes to macrophage tropism of CCR5-using HIV-1 envelopes

While CCR5 is the principal coreceptor used by macrophage (M)-tropic HIV-1, not all primary CCR5-using (R5) viruses enter macrophages efficiently. Here, we used functionally-diverse R5 envelope (Env) clones to characterize virus-cell interactions important for efficient CCR5-mediated macrophage entry. The magnitude of macrophage entry by Env-pseudotyped reporter viruses correlated with increased immunoreactivity of CD4-induced gp120 epitopes, increased ability to scavenge low levels of cell-surface CCR5, reduced sensitivity to the CCR5 inhibitor maraviroc, and increased dependence on specific residues in the CCR5 ECL2 region. These results are consistent with an altered and more efficient mechanism of CCR5 engagement. Structural studies revealed potential alterations within the gp120 V3 loop, the gp41 interaction sites at the gp120 C- and N-termini, and within the gp120 CD4 binding site which may directly or indirectly lead to more efficient CCR5-usage. Thus, enhanced gp120-CCR5 interactions may contribute to M-tropism of R5 HIV-1 strains through different structural mechanisms.

Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain

ABSTRACT Most human immunodeficiency virus type 1 (HIV-1) strains isolated from the brain use CCR5 for entry into macrophages and microglia. Strains that use both CCR5 and CXCR4 for entry (R5X4 strains) have been identified in the brains of some individuals, but mechanisms underlying the persistence of R5X4 viruses compartmentalized between the brain and other tissue reservoirs are unknown. Here, we characterized changes in the HIV-1 envelope (Env) that enhance the tropism of R5X4 variants for brain or lymphoid tissue. R5X4 Envs derived from the brains of two individuals had enhanced CCR5 usage in fusion assays compared to R5X4 Envs derived from matched spleen or blood, which was associated with reduced dependence on specific residues in the CCR5 N terminus and extracellular loop 1 (ECL1) and ECL3 regions. In contrast, spleen/blood-derived Envs had enhanced CXCR4 usage compared to brain-derived Envs, which was associated with reduced dependence on residues in the CXCR4 N terminus and ECL2 region. Consequently, brain-derived Envs had preferential CCR5 usage for HIV-1 entry into the JC53 cell line, could use either CCR5 or CXCR4 for entry into monocyte-derived macrophages (MDM), and could use CCR5 (albeit inefficiently) for entry into peripheral blood mononuclear cells (PBMC), whereas the entry of spleen-derived Envs was CXCR4 dependent in all three cell types. Mutagenesis studies of Env amino acid variants influencing coreceptor usage showed that S306 in the gp120 V3 region of brain-derived Envs reduces dependence on the CCR5 N terminus and enhances CCR5 usage for HIV-1 entry into PBMC and MDM, whereas R306 in spleen-derived Envs reduces dependence on the CXCR4 N terminus and confers the CXCR4 restricted phenotype. These results identify mechanisms underlying R5X4 HIV-1 persistence in different tissue reservoirs. Tissue-specific changes in the gp120 V3 region that increase the efficiency of CCR5 or CXCR4 usage, and thereby influence coreceptor preference, may enhance the tropism of R5X4 strains for CCR5-expressing macrophage lineage cells in the brain and CXCR4-expressing T cells in lymphoid tissues, respectively.

Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net charge.

To better understand the evolution of the viral envelope glycoproteins (Env) in HIV-1 infected individuals who progress to AIDS maintaining an exclusive CCR5-using (R5) virus population, we cloned and sequenced the env gene of longitudinally obtained primary isolates. A shift in the electrostatic potential towards an increased net positive charge was revealed in gp120 of end-stage viruses. Residues with increased positive charge were primarily localized in the gp120 variable regions, with the exception of the V3 loop. Molecular modeling indicated that the modifications clustered on the gp120 surface. Furthermore, correlations between increased Env net charge and lowered CD4(+) T cell counts, enhanced viral fitness, reduced sensitivity to entry inhibitors and augmented cell attachment were disclosed. In summary, this study suggests that R5 HIV-1 variants with increased gp120 net charge emerge in an opportunistic manner during severe immunodeficiency. Thus, we here propose a new mechanism by which HIV-1 may gain fitness.

Genetic and Functional Analysis of R5X4 Human Immunodeficiency Virus Type 1 Envelope Glycoproteins Derived from Two Individuals Homozygous for the CCR5Δ32 Allele

ABSTRACT We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) isolated from two HIV-1-infected CCR5Δ32 homozygotes. Envs from both subjects used CCR5 and CXCR4 for entry into transfected cells. Most R5X4 Envs were lymphocyte-tropic and used CXCR4 exclusively for entry into peripheral blood mononuclear cells (PBMC), but a subset was dually lymphocyte- and macrophage-tropic and used either CCR5 or CXCR4 for entry into PBMC and monocyte-derived macrophages. The persistence of CCR5-using HIV-1 in two CCR5Δ32 homozygotes suggests the conserved CCR5 binding domain of Env is highly stable and provides new mechanistic insights important for HIV-1 transmission and persistence.

Is the central nervous system a reservoir of HIV-1?

Purpose of reviewTo summarize the evidence in the literature that supports the central nervous system (CNS) as a viral reservoir for HIV-1 and to prioritize future research efforts. Recent findingsHIV-1 DNA has been detected in brain tissue of patients with undetectable viral load or neurocognitive disorders, and is associated with long-lived cells such as astrocytes and microglia. In neurocognitively normal patients, HIV-1 can be found at high frequency in these cells (4% of astrocytes and 20% of macrophages). CNS cells have unique molecular mechanisms to suppress viral replication and induce latency, which include increased expression of dominant negative transcription factors and suppressive epigenetic factors. There is also evidence of continued inflammation in patients lacking a CNS viral load, suggesting the production and activity of viral neurotoxins (for example, Tat). SummaryTogether, these findings provide evidence that the CNS can potentially act as a viral reservoir of HIV-1. However, the majority of these studies were performed in historical cohorts (absence of combination antiretroviral therapy or presence of viral load), which do not reflect modern day patients (combination antiretroviral therapy-treated and undetectable viral load). Future studies will need to examine patient samples with these characteristics to conclusively determine whether the CNS represents a relevant and important viral reservoir.

A common mechanism of clinical HIV-1 resistance to the CCR5 antagonist maraviroc despite divergent resistance levels and lack of common gp120 resistance mutations

BackgroundThe CCR5 antagonist maraviroc (MVC) inhibits human immunodeficiency virus type 1 (HIV-1) entry by altering the CCR5 extracellular loops (ECL), such that the gp120 envelope glycoproteins (Env) no longer recognize CCR5. The mechanisms of HIV-1 resistance to MVC, the only CCR5 antagonist licensed for clinical use are poorly understood, with insights into MVC resistance almost exclusively limited to knowledge obtained from in vitro studies or from studies of resistance to other CCR5 antagonists. To more precisely understand mechanisms of resistance to MVC in vivo, we characterized Envs isolated from 2 subjects who experienced virologic failure on MVC.ResultsEnvs were cloned from subjects 17 and 24 before commencement of MVC (17-Sens and 24-Sens) and after virologic failure (17-Res and 24-Res). The Envs cloned during virologic failure showed broad divergence in resistance levels, with 17-Res Env exhibiting a relatively high maximal percent inhibition (MPI) of ~90% in NP2-CD4/CCR5 cells and peripheral blood mononuclear cells (PBMC), and 24-Res Env exhibiting a very low MPI of ~0 to 12% in both cell types, indicating relatively “weak” and “strong” resistance, respectively. Resistance mutations were strain-specific and mapped to the gp120 V3 loop. Affinity profiling by the 293-Affinofile assay and mathematical modeling using VERSA (Viral Entry Receptor Sensitivity Analysis) metrics revealed that 17-Res and 24-Res Envs engaged MVC-bound CCR5 inefficiently or very efficiently, respectively. Despite highly divergent phenotypes, and a lack of common gp120 resistance mutations, both resistant Envs exhibited an almost superimposable pattern of dramatically increased reliance on sulfated tyrosine residues in the CCR5 N-terminus, and on histidine residues in the CCR5 ECLs. This altered mechanism of CCR5 engagement rendered both the resistant Envs susceptible to neutralization by a sulfated peptide fragment of the CCR5 N-terminus.ConclusionsClinical resistance to MVC may involve divergent Env phenotypes and different genetic alterations in gp120, but the molecular mechanism of resistance of the Envs studied here appears to be related. The increased reliance on sulfated CCR5 N-terminus residues suggests a new avenue to block HIV-1 entry by CCR5 N-terminus sulfopeptidomimetic drugs.

Entinostat is a histone deacetylase inhibitor selective for class 1 histone deacetylases and activates HIV production from latently infected primary T cells

Objectives:To compare the potency, toxicity and mechanism of action of multiple histone deacetylase inhibitors (HDACi) in activating HIV production from latency. Design:In-vitro analysis of HDACi in a primary T-cell model of HIV latency and latently infected cell lines. Methods:Latently infected chemokine ligand 19 (CCL19)-treated CD4+ T cells and the latently infected cell lines ACH2 and J-Lat were treated with a panel of HDACi, including entinostat, vorinostat, panonbinostat and MCT3. Viral production and cell viability were compared. Expression of cellular HDACs was measured by western blot and PCR. Association of HDACs with the HIV long-terminal repeat (LTR) using latently infected CCL19-treated primary CD4+ T cells in the presence and absence of specific HDACi was determined by chromatin immunoprecipitation (ChIP). Results:We demonstrated considerable variation in the potency and toxicity of HDACi in latently infected primary CD4+ T cells and cell lines. All HDACi tested activated HIV production in latently infected primary T cells with greatest potency demonstrated with entinostat and vorinostat and greatest toxicity with panobinostat. Following the addition of HDACi in vitro, there were no changes in markers of T-cell activation or expression of the HIV coreceptors chemokine (C-X-C motif) receptor 4 (CXCR4) or chemokine (C-C motif) receptor type 5 (CCR5). ChIP analysis of latently infected CCL19-treated primary CD4+ T cells showed binding by HDAC1, HDAC2 and HDAC3 to the LTR with removal of HDAC1 and HDAC2 following treatment with the HDACi vorinostat and HDAC1 only following treatment with entinostat. Conclusion:The HDACi entinostat, selective for inhibition of class I HDACs, induced virus expression in latently infected primary CD4+ T cells making this compound an attractive novel option for future clinical trials.