GENETICS OF PRIMATE 'D' TYPE RETROVIRUSES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 了解逆转录病毒的功能对于寻求治疗或预防艾滋病毒至关重要。本研究的目的是：（1）确定Gag与细胞骨架运动机制的相互作用，（2）确定病毒和细胞组分在衣壳从组装位点转运到质膜中的作用，（3）确定衣壳与细胞ESCRT机制的相互作用，（4）研究病毒出芽中涉及的肉豆蔻基开关机制。 我们证明了M-PMV MA与动力蛋白轻链Tc-tex 1相互作用。这表明逆转录病毒如何篡夺现有的细胞运输机制，以集中和定位其结构组分，以实现有效的衣壳组装。在M-PMV MA蛋白的单个氨基酸变化后观察到意外的主要结构改变。 这些结果为C型形态发生R55 F突变体的戏剧性表型提供了明确的结构基础，因为结构域转换封闭了潜在的Tc-tex 1结合基序。其他突变的碱性氨基酸残基在MA结构域的Gag显示，残基K16和K20发挥重要作用，限制M-PMV衣壳与膜的相互作用，大概是通过调节挤出的N-末端肉豆蔻酸部分，因为任何一个残基的突变的结果在出芽到最近的囊泡。 这与肉豆蔻酸酯在磷脂酰肌醇触发的质膜挤出后衣壳的膜包裹中发挥关键作用一致。R22 A突变体组装正常，但似乎在皮质肌动蛋白层的运输中被阻止  这表明转运可能是一个两步过程，最初涉及微管，然后涉及肌动蛋白丝。我们已经开发了一种功能性的Gag-GFP表达前病毒，这将使我们能够通过视频显微镜在真实的时间内跟踪衣壳组装和运输。 最后，我们首次证明β逆转录病毒进入细胞可以被新世界猴TRIM-5a蛋白抑制，表明这种细胞限制系统的多样性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Understanding the function of retroviruses is critical in seeking treatment or prevention of HIV. The objective of this study was to: (1) define the interaction of Gag with the cytoskeletal motor machinery, (2) determine the role of viral and cellular components in the transport of capsids from the site of assembly to the plasma membrane, (3) define the interactions of capsids with the ESCRT machinery of the cell, and (4) investigate the myristyl switch mechanism that is involved in virus budding. We demonstrated that M-PMV MA interacts with the dynein light chain Tc-tex1. This shows how retroviruses usurp an existing cellular transport machinery to concentrate and localize their structural components for efficient capsid assembly. Unexpected major structural alterations were observed following single amino acid changes in the M-PMV MA protein. These results provided a clear structural basis for the dramatic phenotype of the C-type morphogenesis R55F mutant since the domain switch occludes the potential Tc-tex1 binding motif. Other mutation of basic amino acid residues in the MA domain of Gag revealed that residues K16 and K20 play an important role in restricting M-PMV capsid interactions with membranes, presumably by regulating extrusion of the N-terminal myristate moiety, since mutation of either residue results in budding into the nearest vesicle. This is consistent with myristate playing a key role in membrane envelopment of the capsid following a phosphoinositol-triggered extrusion at the plasma membrane. The R22A mutant assembles normally but appears to be arrested in transport at the cortical actin layer  suggesting that transport may be a two step process, initially involving microtubules then actin filaments. We have developed a functional Gag-GFP expressing provirus that will allow us to follow capsid assembly and transport in real time by video microscopy. Finally, we showed for the first time that betaretrovirus entry into the cell can be inhibited by new world monkey TRIM-5a proteins, indicating the diversity of this cellular restriction system.