Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

Peer reviewe

Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination

International audienceBackground: Through incorporation into virus particles, the HIV‑1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhance‑ ment of virus infectivity in established CD4‑positive cell lines via a nonenzymatic mechanism. Results: We report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV‑1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte‑derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2‑ and RPA32‑depleted T lymphocytes. Reciprocally, viruses produced in UNG2‑depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination. Conclusions: Our data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV‑1

STAT5 and Oct-1 Form a Stable Complex That Modulates Cyclin D1 Expression

Signal transducer and activator of transcription 5 (STAT5) is activated by numerous cytokines that control blood cell development. STAT5 was also shown to actively participate in leukemogenesis. Among the target genes involved in cell growth, STAT5 had been shown to activate cyclin D1 gene expression. We now show that thrombopoietin-dependent activation of the cyclin D1 promoter depends on the integrity of a new bipartite proximal element that specifically binds STAT5A and -B transcription factors. We demonstrate that the stable recruitment of STAT5 to this element in vitro requires the integrity of an adjacent octamer element that constitutively binds the ubiquitous POU homeodomain protein Oct-1. We observe that cytokine-activated STAT5 and Oct-1 form a unique complex with the cyclin D1 promoter sequence. We find that STAT5 interacts with Oct-1 in vivo, following activation by different cytokines in various cellular contexts. This interaction involves a small motif in the carboxy-terminal region of STAT5 which, remarkably, is similar to an Oct-1 POU-interacting motif present in two well-known partners of Oct-1, namely, OBF-1/Bob and SNAP190. Our data offer new insights into the transcriptional regulation of the key cell cycle regulator cyclin D1 and emphasize the active roles of both STAT5 and Oct-1 in this process

Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single <em>In Vitro</em> Screening System

<div><p>Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such <em>in vivo</em> experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34⁺CD38^−/low(CD45RA⁻CD90⁺) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.</p> </div

T cell differentiation from CD34⁺CD38^−/low cells cultured with MS5 cell lines in presence of 1 µg/ml doxycyclin.

<p>(A) 5000 cells were cultured in complete medium with MS5/DL1 and MS5/DL1^ind cell lines. Flow cytometry was performed on the progeny of such cells 21 and 42 days after initiating the cultures. Shown are results of labelled cells with anti-human specific antibodies directed against CD34, CD7, CD4, CD8 and CD3. % of positive cells are indicated beside each quadrant. (B) Summary results of analysing the progeny of 5–10×10³ CD34⁺CD38^−/low cells from 7–8 individual CB samples. Left: shown are % of CD7⁺ cells and every symbols represent an individual sample. Right, Proportion of indicated populations gated in the CD7⁺ cells for every time point tested. Shown are mean +/− SEM (C) Levels of NOTCH target gene transcripts at 21 days of culture with doxycyclin. Results were normalized over ß2m transcripts levels for each sample. (D) Follow up of T cell differentiation after switching DL1 expression off (MS5 or MS5/DL1^ind – doxycyclin) during culture. Shown are results from 1 out of 2 experiments. Flow cytometry was performed at 3 time points for the measure of CD4 and CD8 expression on human cells. % of positive cells are indicated beside every dot plot. When a specific gate was drawn, % of cells is indicated beside the gate. K&W statistical analysis was used for data of this figure.</p

Multi-lineage differentiation of human CD34⁺CD38^−/low cells in co-cultures with MS5 cell lines.

<p>Sorted cells (10×10³ cells/well) were cultured 21 days without DL1/expressing stromal cells (MS5 or MS5/DL1^ind100–doxycyclin). Cells were harvested and analysed by FACS for the presence of myeloid (CD14⁺/CD15⁺), lymphoid B (CD19⁺) and progenitor (CD34⁺) cells. Half of cells were plated with DL1/expressing stromal cells (MS5/DL1 or MS5/DL1^ind100+ doxycyclin). (A) Results of a representative experiment. % of expressing cells are indicated under the plots in case of quadrant stat or beside the gated population. (B) Summary results of 3 CB samples. (C) Levels of <i>Gata3</i>, <i>pT</i>α, <i>TCF7</i> and <i>Hes1</i> transcripts in the progeny of CD34⁺CD38^−/low cells before (MS5 or MS5/DL1^ind100–doxycyclin) and after (MS5/DL1 or MS5/DL1^ind100+doxycyclin) DL1 induction during culture. Results were normalized over ß2m transcript levels. M&W and K&W statistical analyses were respectively used in (B) and (C).</p