HIV Drug Resistance Program, National Cancer Institute and SAIC-Frederick, NCI-Frederick, Frederick, Maryland, United States of America.
PLoS Pathog. 2010 Feb 26;6(2):e1000788. doi: 10.1371/journal.ppat.1000788.
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.
我们开发了一种高效的方法来量化具有单周期、依赖复制的报告载体的细胞间感染。该系统用于研究 HTLV-1 和 HIV-1 载体在淋巴细胞系中的感染机制。用报告载体、包装载体和 Env 表达质粒转染的效应细胞产生病毒样颗粒，将报告基因活性转导至共培养的靶细胞中，背景为零。仅在靶细胞中检测到报告基因表达，并且需要 Env 表达质粒和病毒包装载体，它们为病毒复制提供必需的结构和酶蛋白。细胞-细胞融合对感染没有贡献，因为在合胞体中很少检测到报告蛋白。与 Raji/CD4 细胞的无细胞感染相比，共培养转染的 Jurkat T 细胞和靶 Raji/CD4 细胞增强了 HIV-1 感染两倍，增强了 HTLV-1 感染一万倍。干扰肌动蛋白和微管聚合的试剂强烈抑制 HTLV-1 并适度降低 HIV-1 细胞间感染，表明细胞骨架重塑对 HTLV-1 传播更为重要。时程研究表明，HTLV-1 传播在细胞混合后很快发生，而 HIV-1 共培养感染的较慢动力学提示了不同的感染传播机制。已经证明 HTLV-1 Tax 在改变增强病毒感染和复制的细胞-细胞相互作用中发挥重要作用。有趣的是，Jurkat 细胞和 Raji/CD4 细胞之间的超抗原诱导的突触并不增强 HTLV-1 或 HIV-1 的感染。一般来说，对细胞间感染的依赖性取决于病毒、效应细胞和靶细胞类型以及细胞-细胞相互作用的性质。
