Dominique Bergeron

Incorporation of Vpr into human immunodeficiency virus type 1: role of conserved regions within the P6 domain of Pr55gag.

The 96-amino acid Vpr protein is the only virion-associated regulatory protein encoded by the human immunodeficiency virus type 1 (HIV-1). Vpr incorporation into the viral particle is most likely due to an interaction with a viral structural protein. Recent data have shown that DNA encoding for the p55 Gag precursor protein (Pr55gag) is the minimal viral genetic information necessary for Vpr incorporation. Other studies have suggested that the p6 portion of Pr55gag, which is unique to lentiviruses, is involved in Vpr incorporation. To investigate the mechanism of incorporation of Vpr into HIV-1 virions, COS-7 cells were cotransfected with ptrENV, an expression vector that encodes all of the HIV-1 regulatory proteins including Rev and Vpr, and different constructs of pIIIgagCAR, a rev-dependent Gag expression plasmid that encodes Pr55gag and the viral protease. Virions produced from gag constructs containing a premature p6 termination codon at positions Leu-1, Ser-17, Tyr-36, or Leu-44 lacked detectable Vpr. In contrast, gag constructs with double Pro-10-Pro-11 substitutions for Leu-10-Leu-11 or a premature termination codon at position Pro-49 of p6 were still able to incorporate Vpr, however, with lower efficiency than wild type. The mutations described in this study affected directly two short regions within the p6 domain, which are highly conserved among primate immunodeficiency viruses. Our results suggest that the conserved (P-T/S-A-P-P) and (L-X-S-L-F-G) motifs located near the N-terminus and C-terminus, respectively, of the p6 domain of Gag are critical for Vpr incorporation into HIV-1 virions.

An improved method for purifying human thymic dendritic cells

Thymic dendritic cells (DC) play a prominent role in the immune response as they constitute a key element involved in the maturation of thymocytes in the thymus. Human thymic DC, like DC from other lymphoid organs, represent a minor cell population (< 2%) of the thymus. Since these cells cannot replicate in vitro, the development of efficient purification methods is an essential prerequisite for extensive functional studies. DC express high levels of HLA-DR, a cell surface marker of the MHC class II antigen which is not exclusive to DC. Since no specific human thymic DC marker has been identified so far, DC purification methods are mainly based on depletion of particular subgroups of cells. We report here an improved method for purifying human thymic dendritic cells. In contrast to prior work, CD2+ thymocytes were first depleted by rosetting with neuraminidase treated sheep red blood cells. The nonrosetted cells were separated in a Percoll gradient, and the low-density cells were subsequently depleted of nondendritic cells by using thymocyte and macrophage specific monoclonal antibodies and either magnetic bead depletion or cytofluorometry. Cell populations (18-55 x 10(6) cells) obtained following magnetic bead purification were at least 80% HLA-DR+/CD2- and exhibited ultrastructural morphological features and functional activities such as those described previously for thymic DC. This improved method was compared with different purification approaches that use various combinations of cell density-based separation techniques and cell surface specific markers antibody reactivity. The magnetic beads depletion approach provided higher yields.

Infection of Human Thymic Dendritic Cells with HIV-1 Induces the Release of a Cytotoxic Factor(s)

Lymphoid organs have been reported to represent the major site for the establishment and the propagation of human immunodeficiency virus (HIV-l) (1). HIV-1 infection is characterized by a permanent high-level of viral replication associated with a high-level turnover of CD4+ T cells (2). The late stages of the disease are characterized by a progressive depletion of CD4+ T cells that eventually lead to the development of the acquired immune deficiency syndrome (AIDS). A variety of direct and indirect mechanisms have been proposed to account for the decline of the CD4+ T cells (3). Among them, inappropriate activation of apoptosis, a programmed cell death process, has been proposed to contribute to the CD4+ T cell depletion (4). HIV-induced apoptotic cell death has been demonstrated not only in vitro but also in infected individuals and is now considered to be a major mechanism of HIV-mediated cell death (5–7).