Pierre Corbeau

Suppression of microRNA-silencing pathway by HIV-1 during virus replication.

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1–infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.

Suv39H1 and HP1γ are responsible for chromatin‐mediated HIV‐1 transcriptional silencing and post‐integration latency

HIV‐1 gene expression is the major determinant regulating the rate of virus replication and, consequently, AIDS progression. Following primary infection, most infected cells produce virus. However, a small population becomes latently infected and constitutes the viral reservoir. This stable viral reservoir seriously challenges the hope of complete viral eradication. Viewed in this context, it is critical to define the molecular mechanisms involved in the establishment of transcriptional latency and the reactivation of viral expression. We show that Suv39H1, HP1γ and histone H3Lys9 trimethylation play a major role in chromatin‐mediated repression of integrated HIV‐1 gene expression. Suv39H1, HP1γ and histone H3Lys9 trimethylation are reversibly associated with HIV‐1 in a transcription‐dependent manner. Finally, we show in different cellular models, including PBMCs from HIV‐1‐infected donors, that HIV‐1 reactivation could be achieved after HP1γ RNA interference.

CD4+ T Cell Surface CCR5 Density as a Determining Factor of Virus Load in Persons Infected with Human Immunodeficiency Virus Type 1

The intensity of expression of the chemokine receptor CCR5 is involved in in vitro cell infectability by human immunodeficiency virus (HIV)-1 R5 isolates. Because CCR5 expression varies among individuals, the hypothesis that this expression could determine virus load in HIV-1-infected persons was tested. The mean number of CCR5 molecules per cell was measured on peripheral blood CD4+ T lymphocytes (CCR5 density) from HIV-1-infected, asymptomatic, nontreated adults. There was a strong correlation between HIV RNA plasma level and CCR5 density (P=.009) that was independent of cell activation and was not due to an HIV-induced CCR5 up-regulation. These data are compatible with the hypothesis that CCR5 density is a key factor governing cell infectability and in vivo virus production and explain the protective effect of the Delta32CCR5 deletion, which results in low CCR5 expression. CCR5 density might be of critical predictive value in HIV infection.

Cell surface CCR5 density determines the postentry efficiency of R5 HIV-1 infection

We have recently reported that the mean number of CCR5 coreceptors at the surface of CD4+ T cells (CCR5 density) correlates with viral load and disease progression in HIV-1-infected persons. Here, we definitively establish that CCR5 density determines the level of virus production and identify the stages of HIV-1 replicative cycle modulated by this effect. We show, by transducing the CCR5 gene into CCR5+ cells, that CCR5 overexpression resulted in an HIV-1 overinfectability. We sorted HOS-CD4+-CCR5+ cells into two subpopulations, HOShigh and HOSlow, the former expressing seven times more cell surface CCR5 molecules than the latter. Virus production was 30–80 times higher in HOShigh cells than in HOSlow cells after a single round of infection. In contrast, only twice as many viral particles entered the cytosol of HOShigh cells as compared with the cytosol of HOSlow cells. Yet, seven times as many early, and 24 times as many late, reverse transcription products were found in HOShigh cells as compared with HOSlow cells. Moreover, a 24- to 30-fold difference in the number of copies of integrated HIV-1 DNA was observed. No difference in HIV-1 LTR activation between the two cell lines was evident. Finally, we show that the higher virus production observed in HOShigh cells is inhibited by pertussis toxin, a Gαi protein inhibitor. Thus, CCR5 density mainly modulates postentry steps of the virus life cycle, particularly the reverse transcription. These data explain why CCR5 density influences HIV-1 disease progression and underline the therapeutic interest of lowering CCR5 expression.

Serum procalcitonin and C-reactive protein concentrations to distinguish mildly infected from non-infected diabetic foot ulcers: a pilot study

Aims/hypothesisInfection of diabetic foot ulcers is common; at early stages it is difficult to differentiate between non-infected ulcers (or those colonised with normal flora) and ulcers infected with virulent bacteria that lead to deterioration. This pilot study aimed to assess the diagnostic accuracy of inflammatory markers as an aid to making this distinction.MethodsWe included 93 diabetic patients who had an episode of foot ulcer and had not received antibiotics during the 6 months preceding the study. Ulcers were classified as infected or uninfected, according to the Infectious Diseases Society of America–International Working Group on the Diabetic Foot classification. Diabetic patients without ulcers (n = 102) served as controls. C-reactive protein (CRP), orosomucoid, haptoglobin and procalcitonin were measured together with white blood cell and neutrophil counts. The diagnostic performance of each marker, in combination (using logistic regression) or alone, was assessed.ResultsAs a single marker, CRP was the most informative for differentiating grade 1 from grade 2 ulcers (sensitivity 0.727, specificity 1.000, positive predictive value 1.000, negative predictive value 0.793) with an optimal cut-off value of 17 mg/l. In contrast, white blood cell and neutrophil counts were not predictive. The most relevant combination derived from the logistic regression was the association of CRP and procalcitonin (AUC 0.947), which resulted in a significantly more effective determination of ulcer grades, as shown by comparing receiver operating characteristic curves.Conclusions/interpretationMeasurement of only two inflammatory markers, CRP and procalcitonin, might be of value for distinguishing between infected and non-infected foot ulcers in subgroups of diabetic patients, to help ensure the appropriate allocation of antibiotic treatment. Nevertheless, external validation of the diagnostic value of procalcitonin and CRP in diabetic foot ulcers is needed before routine use can be recommended.

CD4 T cell surface CCR5 density as a host factor in HIV-1 disease progression.

Objective and designWe have recently shown that the number of CCR5 molecules at the surface of peripheral blood CD4 T cells (CCR5 density) correlates with the viral RNA plasma level in HIV-1-infected individuals. As viral load is a strong predictor of outcome in HIV infection, the present study examines the correlation between CCR5 density and HIV-1 disease progression. MethodsUsing a quantitative flow cytometry assay, we measured CCR5 density in HIV-1-infected adults and control healthy volunteers. The CCR5 genotype (presence of a Δ32 allele) was also determined. ResultsCCR5 density was stable over time on non-activated, HLA-DR−CD4 T cells of infected individuals. In a study cohort of 25 patients, asymptomtic and non-treated, we observed a correlation between CCR5 density on HLA-DR−CD4 T cells and the CD4 T cell slope (P = 0.026), which was independent of the presence or absence of the Δ32CCR5 deletion. In particular, slow progressors expressed lower CCR5 densities than non-slow progressors (P = 0.004) and non-infected control subjects (P = 0.002). ConclusionThese results are compatible with the hypothesis that CCR5 density, which is a key factor of HIV-1 infectability, determines in-vivo HIV production, and thereby the rate of CD4 cell decline. Consequently, CCR5 density quantitation could be a new valuable prognostic tool in HIV-1 infection. Moreover, these data emphasize the therapeutic potential of treatments that reduce functional CCR5 density.

An antibody that binds the immunoglobulin CDR3-like region of the CD4 molecule inhibits provirus transcription in HIV-infected T cells.

We used the polymerase chain reaction (PCR) to study which step(s) of the human immunodeficiency virus type 1 (HIV‐1) life cycle may be blocked following treatment of HIV‐exposed CEM cells with 13B8‐2, a monoclonal antibody (mAb) specific for the immunoglobulin (Ig) CDR3‐like region of the CD4 molecule and able to inhibit the productive infection of CEM cells by HIV‐1. The presence of viral RNA was investigated and found in 13B8‐2 mAb‐treated CEM cells 30 min after viral exposure; the full‐length viral DNA was found at 24 h post‐infection. We also found integrated forms of viral DNA at 24 h post‐infection. However, the integrated provirus was transcriptionally inactive in 13B8‐2 mAb‐treated cells, as demonstrated by the absence of spliced HIV‐1 mRNA. The lack of HIV transcription under 13B8‐2 mAb treatment was confirmed by chloramphenicol acetyltransferase (CAT) assay. We conclude that the inhibition of viral gene transcription accounts for the lack of progeny virions in culture supernatants of cells treated with this anti‐CD4 mAb. We also demonstrate that 13B8‐2 blocks viral production from chronically infected cells and restores CD4 cell‐surface expression on CEM cells containing an integrated provirus(es). We found this effect to be reversible. Moreover, we demonstrate that 13B8‐2 mAb treatment is efficient on different HIV‐1 and HIV‐2 virus isolates. These results may have major implications for the treatment of AIDS.

Small-Molecule Inhibition of HIV pre-mRNA Splicing as a Novel Antiretroviral Therapy to Overcome Drug Resistance

The development of multidrug-resistant viruses compromises antiretroviral therapy efficacy and limits therapeutic options. Therefore, it is an ongoing task to identify new targets for antiretroviral therapy and to develop new drugs. Here, we show that an indole derivative (IDC16) that interferes with exonic splicing enhancer activity of the SR protein splicing factor SF2/ASF suppresses the production of key viral proteins, thereby compromising subsequent synthesis of full-length HIV-1 pre-mRNA and assembly of infectious particles. IDC16 inhibits replication of macrophage- and T cell–tropic laboratory strains, clinical isolates, and strains with high-level resistance to inhibitors of viral protease and reverse transcriptase. Importantly, drug treatment of primary blood cells did not alter splicing profiles of endogenous genes involved in cell cycle transition and apoptosis. Thus, human splicing factors represent novel and promising drug targets for the development of antiretroviral therapies, particularly for the inhibition of multidrug-resistant viruses.

Maraviroc intensification of stable antiviral therapy in HIV-1-infected patients with poor immune restoration: MARIMUNO-ANRS 145 study.

Objective:To address the ability of a 24-week Maraviroc (MVC) intensification of a stable antiretroviral therapy (cART) to significantly increase the CD4 cell count slope. Methods:Patients were eligible if they had CD4 <350 cells/mm3, a CD4 slope <50 cells/mm3 per year, and sustained plasma HIV-RNA <50 copies/mL over the last 2 years, while receiving a stable cART. Patients harboring pure X4-using viruses by a phenotypic tropism assay were excluded. MVC was added to cART for 24 weeks, at the recommended dosage per drug–drug interactions. The primary endpoint was a significant positive difference in CD4 slopes (with MVC— pre-MVC, paired t test). Results:Sixty patients (55 men), with median age 51 years, baseline CD4 238 cells/mm3, and slope before intensification +14.1 cells/mm3 per year were included. CD4 nadir was <50/mm3 in 47% of the population. The full set of patients (N = 57) completed week 24, and the on-treatment patients (N = 48) did not discontinue MVC. The median CD4 slope difference from baseline was +22.6 cells/mm3 per year (P = 0.08) in full set and +22.6 cells/mm3 per year (P = 0.04) in on-treatment. Slope evolution was not different according to baseline tropism, CD4 nadir, or ongoing cART regimen. No drug-related severe adverse events were recorded during intensification. MVC plasma concentrations were significantly different depending on drug–drug interaction with ongoing cART regimen and tended to be correlated with CD4 cells increase. Conclusion:In this study, MVC intensification of stable cART over 24 weeks was able to enhance CD4 cell slopes in patients with prior insufficient immune restoration despite long-term virological control.

CCR5 antagonism in HIV infection: ways, effects, and side effects

New drugs are needed to improve the convenience of anti-HIV therapy, to reduce its toxicity, and to enhance its activity against both wild-type and resistant viral strains. A number of promising compounds are in development both in already existing classes (reverse transcriptase, protease, and fusion inhibitors) and in new classes (integrase inhibitors). Yet, the identification of drugs that exploit new targets in HIV life cycle remains an important therapeutic objective. The discovery that the C-C chemokine receptor CCR5 is the main coreceptor for HIV-1 [1–5], in addition to the CD4 receptor, has opened new therapeutic possibilities. Thus, the first CCR5 inhibitor, maraviroc, has been approved for use in HIV-infected individuals in 2007. In this article, we will briefly review the biology of CCR5, discuss the virological and immunological consequences of blocking CCR5, and present alternative strategies to neutralize this coreceptor.