Christine Chable-Bessia

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi–Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

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.

SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+T-cells

BackgroundQuiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription.ResultsExamination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment.ConclusionHere, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.

A non-proteolytic role for ubiquitin in Tat-mediated transactivation of the HIV-1 promoter.

The human immunodeficiency virus type 1 (HIV-1) encodes a potent transactivator, Tat, which functions through binding to a short leader RNA, called transactivation responsive element (TAR). Recent studies suggest that Tat activates the HIV-1 long terminal repeat (LTR), mainly by adapting co-activator complexes, such as p300, PCAF and the positive transcription elongation factor P-TEFb, to the promoter. Here, we show that the proto-oncoprotein Hdm2 interacts with Tat and mediates its ubiquitination in vitro and in vivo. In addition, Hdm2 is a positive regulator of Tat-mediated transactivation, indicating that the transcriptional properties of Tat are stimulated by ubiquitination. Fusion of ubiquitin to Tat bypasses the requirement of Hdm2 for efficient transactivation, supporting the notion that ubiquitin has a non-proteolytic function in Tat-mediated transactivation.

Intrinsic ubiquitination activity of PCAF controls the stability of the oncoprotein Hdm2

The p300–CBP-associated factor (PCAF) is a histone acetyltransferase (HAT) involved in the reversible acetylation of various transcriptional regulators, including the tumour suppressor p53. It is implicated in many cellular processes, such as transcription, differentiation, proliferation and apoptosis. We observed that knockdown of PCAF expression in HeLa or U2OS cell lines induces stabilization of the oncoprotein Hdm2, a RING finger E3 ligase primarily known for its role in controlling p53 stability. To investigate the molecular basis of this effect, we examined whether PCAF is involved in Hdm2 ubiquitination. Here, we show that PCAF, in addition to its acetyltransferase activity, possesses an intrinsic ubiquitination activity that is critical for controlling Hdm2 expression levels, and thus p53 functions. Our data highlight a regulatory crosstalk between PCAF and Hdm2 activities, which is likely to have a central role in the subtle control of p53 activity after DNA damage.

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.

HIV-1 Tat targets Tip60 to impair the apoptotic cell response to genotoxic stresses

HIV‐1 transactivator Tat uses cellular acetylation signalling by targeting several cellular histone acetyltransferases (HAT) to optimize its various functions. Although Tip60 was the first HAT identified to interact with Tat, the biological significance of this interaction has remained obscure. We had previously shown that Tat represses Tip60 HAT activity. Here, a new mechanism of Tip60 neutralization by Tat is described, where Tip60 is identified as a substrate for the newly reported p300/CBP‐associated E4‐type ubiquitin‐ligase activity, and Tat uses this mechanism to induce the polyubiquitination and degradation of Tip60. Tip60 targeting by Tat results in a dramatic impairment of the Tip60‐dependent apoptotic cell response to DNA damage. These data reveal yet unknown strategies developed by HIV‐1 to increase cell resistance to genotoxic stresses and show a role of Tat as a modulator of cellular protein ubiquitination.

Competition for XPO5 binding between Dicer mRNA, pre-miRNA and viral RNA regulates human Dicer levels

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function by regulating gene expression post-transcriptionally. Alterations in miRNA expression can strongly influence cellular physiology. Here we demonstrated cross-regulation between two components of the RNA interference (RNAi) machinery in human cells. Inhibition of exportin-5, the karyopherin responsible for pre-miRNA export, downregulated expression of Dicer, the RNase III required for pre-miRNA maturation. This effect was post-transcriptional and resulted from an increased nuclear localization of Dicer mRNA. In vitro assays and cellular RNA immunoprecipitation experiments showed that exportin-5 interacted directly with Dicer mRNA. Titration of exportin-5 by overexpression of either pre-miRNA or the adenoviral VA1 RNA resulted in loss of Dicer mRNA–exportin-5 interaction and reduction of Dicer level. This saturation also occurred during adenoviral infection and enhanced viral replication. Our study reveals an important cross-regulatory mechanism between pre-miRNA or viral small RNAs and Dicer through exportin-5.

ERK5 Activates NF-κB in Leukemic T Cells and Is Essential for Their Growth In Vivo

MAPK cascades play a central role in the cellular response to the environment. The pathway involving the MAPK ERK5 mediates growth factor- and stress-induced intracellular signaling that controls proliferation or survival depending upon the cell context. In this study, we show that reducing ERK5 levels with a specific small hairpin RNA 5 (shERK5) reduced cell viability, sensitized cells to death receptor-induced apoptosis, and blocked the palliative effects of phorbol ester in anti-Fas Ab-treated cells. shERK5 decreased nuclear accumulation of the NF-κB p65 subunit, and conversely, ectopic activation of ERK5 led to constitutive nuclear localization of p65 and increased its ability to trans activate specific reporter genes. Finally, the T lymphoma cell line EL-4, upon expression of shERK5, proliferated in vitro, but failed to induce s.c. tumors in mice. Our results suggest that ERK5 is essential for survival of leukemic T cells in vivo, and thus represents a promising target for therapeutic intervention in this type of malignancy.