The modeled structure of the RNA dependent RNA polymerase of GBV-C Virus suggests a role for motif E in Flaviviridae RNA polymerases

BACKGROUND: The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp) plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV) is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV) is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C). Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. RESULTS: We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. CONCLUSION: Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA polymerases

Brucella beta 1,2 cyclic glucan is an activator of human and mouse dendritic cells

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella beta 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella beta 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella beta 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies

HIV-1 is stored by follicular dendritic cells in lymph nodes even under antiviral treatments

International audienc

Letter to the Editor

International audienc

Un monde de virus: Le mot des coordinatrices

International audienceNo abstract availabl

Quantitative Analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) Infection Using Co-Culture with Jurkat LTR-Luciferase or Jurkat LTR-GFP Reporter Cells

International audienceUnlike HIV-1, HTLV-1 viral transmission requires cell-to-cell contacts, while cell-free virions are poorly infectious and almost absent from body fluids. Though the virus uses three nonexclusive mechanisms to infect new target cells: (1) MTOC polarization followed by formation of a virological synapse and viral transfer into a synaptic cleft, (2) genesis of a viral biofilm and its transfer of embedded viruses, or (3) HTLV-1 transmission using conduits. The Tax transactivator and the p8 viral proteins are involved in virological synapse and nanotube formation respectively.HTLV-1 transcription from the viral promoter (i.e., LTR) requires the Tax protein that is absent from the viral particle and is expressed after productive infection. The present chapter focuses on a series of protocols used to quantify HTLV-1 de novo infection of target cells. These techniques do not discriminate between the different modes of transmission, but allow an accurate measure of productive infection. We used cell lines that are stably transfected with LTR-GFP or LTR-luciferase plasmids and quantified Green Fluorescent Protein expression or luciferase activity, since both of them reflect Tax expression

Viral Source-Independent High Susceptibility of Dendritic Cells to Human T-Cell Leukemia Virus Type 1 Infection Compared to That of T Lymphocytes

International audienceHuman T-cell leukemia virus type 1 (HTLV-1)-infected CD4(+) T cells and dendritic cells (DCs) are present in peripheral blood from HTLV-1 carriers. While T-cell infection requires cell-cell contact, DCs might be infected with cell-free virus, at least in vitro. However, a thorough comparison of the susceptibilities of the two cell types to HTLV-1 infection using cell-associated and cell-free viral sources has not been performed. We first determined that human primary monocyte-derived dendritic cells (MDDCs) were more susceptible to HTLV-1 infection than their autologous lymphocyte counterparts after contact with chronically infected cells. Next, a comparison of infection efficiency using nonconcentrated or concentrated supernatants from infected cells as well as purified viral biofilm was performed. Integrated provirus was found after exposure of MDDCs or primary lymphocytes to viral biofilm but not to a viral supernatant. Using a large series of primary cell samples (n = 21), we demonstrated a higher proviral load in MDDCs exposed to viral biofilm than in lymphocytes. This higher susceptibility is correlated to a higher expression of neuropilin-1 on MDDCs than on autologous activated T lymphocytes. Moreover, we show that MDDCs infected with viral biofilm can transmit the virus to lymphocytes. In conclusion, MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro, supporting a model in which DC infection might represent an important step during primo-infection in vivo. IMPORTANCE: HTLV-1 is able to infect several cell types, but viral DNA is mainly found in T lymphocytes in vivo. This supports a model in which T lymphocytes are the main target of infection. However, during the primo-infection of new individuals, incoming viruses might first encounter dendritic cells (DCs), the specialized immune cells responsible for the antiviral response of the host. HTLV-1 cell-free purified viruses can infect dendritic cells in vitro, while T-cell infection is restricted to cell-to-cell transmission. In order to understand the sequence of HTLV-1 dissemination, we undertook a direct comparison of the susceptibilities of the two cell types using cell-associated and cell-free viral sources. We report here that MDDCs are more susceptible to HTLV-1 infection than autologous lymphocytes in vitro and are able to efficiently transmit the virus to lymphocytes. Our results suggest that DCs may represent a true viral reservoir, as the first cell type to be infected in vivo

HTLV-1, the Other Pathogenic Yet Neglected Human Retrovirus: From Transmission to Therapeutic Treatment

Going back to their discovery in the early 1980s, both the Human T-cell Leukemia virus type-1 (HTLV-1) and the Human Immunodeficiency Virus type-1 (HIV-1) greatly fascinated the virology scene, not only because they were the first human retroviruses discovered, but also because they were associated with fatal diseases in the human population. In almost four decades of scientific research, both viruses have had different fates, HTLV-1 being often upstaged by HIV-1. However, although being very close in terms of genome organization, cellular tropism, and viral replication, HIV-1 and HTLV-1 are not completely commutable in terms of treatment, especially because of the opposite fate of the cells they infect: death versus immortalization, respectively. Nowadays, the antiretroviral therapies developed to treat HIV-1 infected individuals and to limit HIV-1 spread among the human population have a poor or no effect on HTLV-1 infected individuals, and thus, do not prevent the development of HTLV-1-associated diseases, which still lack highly efficient treatments. The present review mainly focuses on the course of HTLV-1 infection, from the initial infection of the host to diseases development and associated treatments, but also investigates HIV-1/HTLV-1 co-infection events and their impact on diseases development

Comparative infection of autologous primary T cells and monocytes derived DCs using cell-free virus preparation, viral biofilm, pseudotyped-virus or viral synapse

International audienc

Intercellular transmission of HTLV-1: not all mechanisms have been revealed

International audienceHTLV-1 is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL) and of HTLV-1-Associated Myelopathy/tropical spastic paraparesis (HAM/TSP). It is mainly detected in CD4+ lymphocytes in vivo, but proviral genomes have also been detected although less frequently, in CD8+ T lymphocytes, B lymphocytes, monocytes, macrophages, dendritic cells and other non-lymphoid cells. Virus spread is highly dependent on cell-cell contact. This mode of transmission is correlated with an increased ability of infected cells to migrate, a property linked to cytoskeleton reorganization induced by the viral Tax protein. Cell-to-cell transmission relies on at least three non-exclusive molecular pathways. First, a specialized area, the "virological synapse'' (VS) promotes direct transmission of budding HTLV-1 particles into a synaptic cleft formed between infected and uninfected cells. Second, HTLV-1 particles accumulate at the plasma membrane of infected cells in a biofilm-like extracellular viral assembly that resembles a bacterial biofilm. Viral biofilm is rapidly transmitted to uninfected cells when infected cells contact target cells. Finally, membrane extensions called inter-cellular conduits facilitate HTLV-1 proteins transfer from infected to uninfected target cells, and may stabilize cell-cell contacts. The aim of this review is to summarize the molecular mechanisms of these HTLV-1 transmission pathways