Interplay between HIV-1 replication and RNAi effectors

International audiencen.

Suppression of HIV-1 replication by microRNA effectors

The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART

Flt3L-Mediated expansion of plasmacytoid dendritic cells suppresses HIV infection in humanized mice

Plasmacytoid dendritic cells (plasmacytoid DC, pDC) are major IFN-I producers and have been shown to be affected by HIV through ill-defined mechanisms. In this study, we directly assess the role of pDC in early infection, evaluating whether modulating their abundance can alter viral replication. First, HIV infection of humanized mice induces systemic depletion of pDC, and in the presence of soluble FMS-like tyrosine kinase 3 ligand (Flt3L), pDC levels remain elevated. Flt3L significantly delays the onset of viremia and reduces viral replication via a process that is dependent on pDC and mediated through an enhanced early IFN-I response. pDC from Flt3L-treated mice are more prone to express IFN-a following TLR7 stimulation, but this propensity is gradually decreased during infection. In conclusion, maintaining pDC levels and function is key to effective early viral control, and in this context, these findings provide practical insights for anti-HIV strategies and vaccine design

A hybrid population-based algorithm for the bi-objective quadratic multiple knapsack problem

International audienceIn this paper, the bi-objective quadratic multiple knapsack problem is tackled with a hybrid population-based method. The proposed method starts by computing two reference solutions, where a specialized powerful mono-objective algorithm is used. From both reference solutions, a starting population is built by using a series of perturbations around the solutions. Next, the so-called non-sorting genetic process is combined with a new drop/rebuild operator for generating a series of populations till converging toward an approximate Pareto front with high density. The performance of the hybrid population based algorithm (namely HBPA) is evaluated on a set of benchmark instances of the literature containing both medium and large-scale instances. Its provided results are compared to those achieved by the best methods available in the literature. Encouraging results have been obtained

Hyperthermia stimulates HIV-1 replication.

International audienceHIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42-45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38-40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity

Hyperthermia does not stimulate viral fusion and reverse transcription.

<p>A: Viral fusion assay. One representative experiment is shown. Jurkat cells were exposed for 2 hours at 37°C or 39.5°C to 5 or 100 ng Gag p24/10⁵ cells/0.1 mL of either WT, or a non fusogenic <i>env</i> mutant (F552Y) NL4-3, both bearing the chimeric protein β-lactamase-Vpr. Viral access to the cytoplasm was assessed by flow cytometry, using the ability of β-lactamase to cleave CCF2-AM, a fluorogenic substrate. B: Mean ± SD of 3 independent experiments. C: In vitro assessment of RT activity. Mean ± SD of 2 independent experiments is shown. HIV-1 NL4-3 virions were lysed and incubated at 37°C or 39.5°C for 1 h or 3 h. RT activity was measured with the Innovagen RetroSys RT Activity Kit (405 nm OD). D: Viral DNA synthesis in Jurkat cells. Mean ± SD of 3 independent experiments is shown. Jurkat cells were infected with 50 ng or 250 ng Gag p24 of Δ<i>env</i>(VSV)//mL/10⁶ cells for 2 hours at 37°C or 39.5°C, washed, and grown at 37°C or 39.5°C. 8 hours p.i., cells were harvested and viral DNA was measured by quantitative PCR. As a control, the RT inhibitor nevirapine (NVP) was added during infection.</p

Hyperthermia increases viral reactivation in J-Lat 10.6 cells.

<p>A: Experimental outline. J-Lat 10.6 are Jurkat-derived cells carrying a latent, integrated, provirus, encoding <i>gfp</i> instead of <i>nef</i>. Stimulation of J-Lat 10.6 cells with TNFα triggers HIV-1 reactivation and GFP expression. B: Reactivation of J-Lat 10.6 cells with various stimuli. One representative experiment is shown. J-Lat 10.6 cells were treated with either TNFα, supernatants from IL-2 treated or PHA-activated PBMCs, or co-cultivated with IL-2-treated PBMCs for 48 hours at 37°C or 39.5°C. Viral reactivation was assessed by measuring GFP levels by flow cytometry. C: Mean ± SD of 4 independent experiments. Statistical significance was assessed by a paired t test. p<0.05(*).</p

Role of Hsp90 during HIV-1 transcription and replication.

<p>A: Visualization of HIV transcription sites in U2OS HIVexo cells. One representative cell (at 37°C), displaying a co-localization of Hsp90 and nascent HIV RNA (followed with a YFP-MS2nls reporter protein) is shown. 2.5×10⁵ U2OS HIVexo cells were plated on coverslips and grown overnight at 37°C. Cells were transfected with 50 ng of Tat and 300 ng of YFP-MS2nls. After 4 hours, cells were washed and grown overnight at 37°C or 39.5°C. B: Mean ± SD of 3 independent experiments. Cells showing a transcription spot were scored for their co-localization with Hsp90. The percentage corresponds to cells in which HIV transcription sites co-localize with Hsp90. Statistical significance was assessed by an unpaired t test p<0.05(*). C: effect of the Hsp90 inhibitor 17-AAG on single-cycle HIV infection in P4C5 cells. Mean ± SD of 4 independent experiments is shown. 2×10⁵ P4C5 cells were infected with 30 ng Gag p24 of Δ<i>env</i>(VSV) for 2 hours at 37°C or 39.5°C, in presence of the indicated doses of 17-AAG. Cells were washed and grown at 37°C or 39.5°C at the indicated doses of 17-AAG. Cells were harvested 24 hours p.i. and Gag levels were assessed by flow cytometry. As a negative control, we used DMSO at a concentration corresponding to 250 nM 17-AAG.</p

Cell growth and expression of Hsp70, Hsp90, and surface molecules in hyperthermic T cells.

<p>A: Primary CD4+ T cells were grown at 37°C or 39.5°C for 3 days. Cell growth was assessed every day by direct counting of living cells (Trypan blue exclusion). B: Primary CD4+ T cells were incubated 8 hours at 37°C or 39.5°C. Cell lysates were collected and probed by Western Blot for Hsp90, Hsp70 and Actin. C: Primary CD4+ T cells were grown at 37°C (blue line) or 39.5°C (red line) for 3 days. Cells were stained for the indicated surface markers and fixed in PFA. Isotype-matched monoclonal antibodies were used as negative controls (grey line).</p