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.
Highlights
Retroviruses can infect cells as cell-free particles or by cell-tocell transmission [1,2,3,4,5]
While we suspect that these images represent virus infection, it has been difficult to accurately quantify virus replication and provirus formation in most cell-to-cell infection experiments
Retroviral vectors that encode reporter proteins have been invaluable tools for analyzing retrovirus replication and restriction, but they have had limited utility in cell-to-cell infection studies due to high background noise resulting from reporter expression in the producer cells
Summary
Retroviruses can infect cells as cell-free particles or by cell-tocell transmission [1,2,3,4,5]. In the latter route of infection, specific cell-cell contacts may strongly enhance virus infection by triggering the reorganization of cytoskeletal and cell-surface protein networks to focus virus release toward clustered receptors on an apposed target cell [2,6,7,8,9,10,11,12]. HTLV-1 is a highly cell-associated virus that is most likely disseminated by cell-to-cell transmission in vivo [13]. HIV-1 infection has been studied intensively, and in vitro systems have been used to examine cell-to-cell transmission of virus from infected T-cells or infected macrophages to uninfected T-cells and epithelial cells [2,6,7,8,9,10,11,12,16,17,18], as well as the special situation where
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