Quantitative Comparison of HTLV-1 and HIV-1 Cell-to-Cell Infection with New Replication Dependent Vectors

Abstract

We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

Monoclonal antibody profiling of cell surface proteins associated with the viral biofilms on HTLV-1 transformed cells

“…The Jurkat E6-1 cells were transduced with lentiviral vector encoding Tax essentially as described (Mazurov et al, 2010). Briefly, 293T cells were cotransfected with three plasmids: transfer vector pUCHR-Tax-IRES-GFP (Mazurov et al, 2010) encoding wild type (Wt) HTLV-1 Tax, IRES, and green fluorescent protein (GFP) gene under CMV promoter; HIV-1 packaging plasmid pCMV∆8.2R (Addgene); and pCMV VSVG vector (Addgene) encoding env from vesicular stomatitis virus (VSV) at a 6:4:0.5 ratio, respectively. Cells were transfected overnight with Lipofectamine 2,000 reagent (Invitrogen) according to the manufacturer's instructions.…”

“…Particularly, the initiation and elongation of viral transcription and leukemic transformation of infected T lymphocytes are widely recognized functions of Tax (de la Fuente and Kashanchi, 2004). Tax enhances cell-to-cell transmission of HTLV-1 by the mechanisms of cytoskeleton reorganization, induction of ICAM-1 expression and cell adhesion, and likely by other not yet recognized mechanisms (Mazurov et al, 2010). It is possible that Tax is also involved in VB-mediated cell-to-cell transmission of HTLV-1, therefore, we aimed to find the expression of which cell surface proteins depends on Tax.…”

Transmission intercellulaire de HTLV-1

“…Un second changement de conformation permet l'interaction de la tyrosine 114 de la SU avec GLUT-n'a été observé après une transfusion de produits sanguins acellulaires. In vitro, et à l'exception des cellules dendritiques [8], l'infection des cellules cibles n'est possible qu'après un contact étroit avec une cellule infectée de façon productive [18], ce qui permet d'augmenter d'un facteur 10 000 l'efficacité de l'infection [19]. Ce contact cellule-cellule, parfois appelé « conjugué », est associé à plusieurs événements : migration des cellules infectées vers leur cible, formation d'une synapse, polarisation du centre organisateur des microtubules (microtubule organizing center, MTOC) dans la cellule infectée, et transfert polarisé et localisé du virus au point de contact.…”

“…Ce contact cellule-cellule, parfois appelé « conjugué », est associé à plusieurs événements : migration des cellules infectées vers leur cible, formation d'une synapse, polarisation du centre organisateur des microtubules (microtubule organizing center, MTOC) dans la cellule infectée, et transfert polarisé et localisé du virus au point de contact. Ces évènements dépendent tous d'une réorganisation du cytosquelette qui fait intervenir les réseaux d'actine et de tubuline dans la cellule infectée [19,20]. Ce phénomène est contrôlé par la protéine virale Tax, qui colocalise partiellement avec le MTOC [20].…”

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

“…While Jurkat T cells have been used to study T cell immune interactions, such as virological synapses [20,27,28], even these will quickly settle and begin to stick to cover glass surfaces, eliminating the cellular mobility seen in vivo. OTs can be used both to prevent contact with the cover glass and to manipulate cells to initiate contact and increase cellular density within a FOV.…”