8/17/2023 0 Comments Vector borne transmission htlv1![]() ![]() ![]() ( C) Time course of HIV-1 coculture infection was examined by blocking virus entry and infection as in (B) except that anti-CD4 mAb SIM.2 (open circles) was used to block entry and non-blocking anti-CD4 mAb SIM.4 was used as control (filled circles). For control infections, human plasma from HIV-1-infected patients was used (open squares). Luc activity was measured 48 h after the start of coculture (0 h). ( B) The time course of HTLV-1 coculture infection was examined by blocking virus entry and infection with HTLV-1-infected human plasma (filled squares) at the indicated times after mixing transfected Jurkat cells and Raji/CD4 cells. Luc activity is normalized relative to the amount of viral Gag protein in the supernatants. Raji/CD4 cells were infected with cell-free VLPs produced from transfected 293T cells or by coculture with transfected Jurkat cells. ( A) Comparison of cell-free (CF) and coculture (CC) infection. Graphs represent the mean of at least three independent experiments with error bars indicating standard deviations. Luc activity was not detected in activated human CD4 + T cells from different donors in repeated experiments. Luc activity in the target cells was assayed 48 h after cell mixing. ( B) Jurkat cells were transfected with HTLV-1 packaging plasmid and HTLV1-inLuc reporter vector and then incubated with an equal number of the indicated cells. Cotransfection of the viral vectors with a dominant-negative VPS4A plasmid (VPSdn), which inhibits virus budding, or addition of the reverse transcriptase inhibitor azidotimidine (AZT 20 µm) inhibited Luc transduction. In other cocultures, Jurkat cells transfected with reporter vectors but without Env-expression plasmid (no Env) or without packaging plasmid (no pack) produced no Luc activity above background. The level of infection between Jurkat cells was determined in cultures without Raji/CD4 cells (no Raji). Coculture of Raji/CD4 cells with Jurkat cells transfected with wild type vectors is designated as control. Infection is expressed as relative light units (RLU) of luciferase activity, measured 48 h after cell mixing. ( A) HTLV1-inLuc and HIV1-inLuc vectors were transfected into Jurkat cells with respective packaging vectors and Env expression plasmids transfected Jurkat cells were combined with an equal number of Raji/CD4 cells 24 h later. 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. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. 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. 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. 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. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. 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. 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. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. ![]()
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