GENETICS OF PRIMATE 'D' TYPE RETROVIRUSES

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 目的：确定(1)M-PMV Gag与细胞骨架运动机制的相互作用，(2)病毒和细胞成分在将衣壳从其组装部位运送到质膜中的作用，(3)衣壳与细胞ESCRT机制的相互作用，以及(4)参与萌发的多花柱开关机制。有效地将预先组装的衣壳运输到细胞膜需要一个功能性的囊泡运输途径和包膜(Env)糖蛋白。转运可能发生在由Env或其他病毒成分诱导的管状-囊泡通路上，或者这些相互作用可能启动沿着其他运输网络的Gag转运。我们发现GAG以CTRS依赖的方式与细胞Tctex-1相互作用，Tctex-1是动力蛋白运动复合体的轻链，表明微管依赖的逆行运输到中心周围区域进行衣壳组装。微管也可以解释预先组装的衣壳向质膜的顺行运输。在M-PMV感染的CMMT细胞中，脉冲追逐试验显示，与未经处理的对照组相比，诺康唑诱导的病毒产生延迟了1小时。肌动蛋白的中断导致病毒粒子释放的轻微延迟，而如果中断的话影响可以忽略不计。活细胞成像显示，在微管破裂后，含有GFP标记的GAG的衣壳的线性运动最初减少，随后速度恢复。诺考达唑治疗导致Env转运停止，释放的病毒粒子中Env掺入不足。重要的是，从缺乏所有三种功能细胞骨架成分的细胞中观察到病毒粒子的持续释放，尽管是延迟的，这表明了一种细胞骨架无关的运输方式。替代运输途径的研究正在进行中。这些研究可能会对艾滋病毒/艾滋病的研究产生影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objectives: define (1) M-PMV Gag interactions with the cytoskeletal motor machinery, (2) role of viral and cellular components in transporting capsids from their assembly site to the plasma membrane, (3) capsid interactions with the cellular ESCRT machinery, and (4) the myristyl switch mechanism involved in budding. Efficient transport of pre-assembled capsids to the cell membrane requires a functional vesicular trafficking pathway and envelope (Env) glycoprotein. Transport may occur along a tubular-vesicular pathway induced by Env or another viral component, or these interactions may initiate Gag transport along other trafficking networks. We show Gag interacts in a CTRS-dependent manner with cellular Tctex-1, a light chain of the dynein motor complex, suggesting a microtubule-dependent retrograde transport to the pericentriolar region for capsid assembly. Microtubules might also explain anterograde transport of pre-assembled capsids to the plasma membrane. In M-PMV-infected CMMT cells, pulse-chase assays revealed a nocodazole induced 1-hour delay in virus production compared to untreated controls. Actin disruption led to a minor delay in virion release, while IF disruption had negligible effects. Live cell imaging showed that following microtubule disruption, an initial reduction in linear movement of capsids containing GFP-tagged Gag occurred, followed by a recovery of velocities. Nocodazole treatment led to a cessation in Env transport and deficient Env incorporation in released virions. Importantly, a continued albeit delayed release of virions was observed from cells lacking all three functional cytoskeleton components, suggesting a cytoskeleton-independent mode of transport. Studies are underway to alternate transport pathways. These studies could have implications for HIV/AIDS research.