STRUCTURE/FUNCTION OF HIV/SIV ENVELOPE TRANSMEMBRANE GLYCOPROTEIN GP41

背景：人类免疫缺陷病毒（HIV）和相关猿类免疫缺陷病毒（SIV）的包膜糖蛋白被合成为gp 160前体，其被加工成两种非共价结合的糖蛋白：gp 120和gp 41。gp 120通过与细胞受体CD 4和趋化因子辅助受体结合来介导病毒进入宿主细胞，这两种受体都位于宿主细胞表面。这种结合诱导跨膜gp 41的构象变化，这有利于病毒和宿主膜之间的膜融合。在分子水平上理解这些过程可能会导致抑制HIV感染的直接手段。由于HIV pg 41和密切相关的SIV gp 41是高度糖基化的跨膜蛋白，因此确定它们的高分辨率结构是一个非常困难的问题。在一个渐进的方法中，我们正在研究gp 41的（非糖基化的）功能结构域的结构。gp 41最重要的区域是位于病毒膜外表面的胞外域，其直接介导膜融合事件。HIV和SIV gp 41胞外域都已在大肠杆菌中表达。对于SIV gp 41，NMR和X-射线结构都已得到解决。结果：确定了SIV gp 41胞外域的高分辨率X射线（Hyde/Wang）和NMR（Clore/Gronenborn/Caffrey）结构。通过这两种方法确定的结构是棒状三聚体，其包含在中心组装为卷曲螺旋的三个平行的N-末端α-螺旋，其中三个反平行的C-末端α-螺旋包装在外部，具有连接内螺旋和外螺旋的高度柔性的环。连接内螺旋和外螺旋的环区域，尽管在X射线模型中没有清楚地分辨，但已经在NMR结构中建模，从而允许建立内螺旋和外螺旋之间的连接性。该区域似乎是导致聚集体形成的三聚体在生理pH值下的低溶解度的原因。我们最近的工作使我们假设这些gp 41的高分子聚集体是HIV相关的神经损伤和痴呆的原因。gp 41蛋白的其他功能区域已经在细菌中表达，包括细胞质结构域，其与HIV基质蛋白（核衣壳的组分）以及各种宿主细胞因子相互作用。意义和未来发展方向：gp 41胞外域的高分辨率结构测定将为新型肽抑制剂的设计提供更合理的方法。基于gp 41的物理性质，已经提出了涉及部分缔合蛋白质复合物的膜融合模型。这些模型也可能为靶向gp 41提供有用的线索。更完整的膜融合图像需要对gp 41进行更广泛的结构研究，特别是那些参与介导与gp 120相互作用的区域。各种工程gp 120蛋白的表达，包括被认为与gp 41相互作用的N-和C-末端结构域，已经表达并正在测试中。摘要：人类免疫缺陷病毒（HIV）表面上的包膜蛋白介导病毒进入其宿主细胞，已在细菌中使用重组DNA方法表达。对囊膜蛋白进行了纯化和结构研究。希望对这些蛋白质的分子细节的理解，对病毒生命周期至关重要，将有助于开发阻断其功能的试剂和方法。- HIV/SIV，囊膜糖蛋白gp 41，膜融合，重组蛋白表达

Background:The envelope glycoprotein of the human (HIV) and related simian (SIV) immunodeficiency virus are synthesized as gp160 precursors which are processed into two non-covalently associated glycoproteins: gp120 and gp41. The gp120 mediates viral entry into the host cell by binding to the cellular receptor CD4 and a chemokine coreceptor, both of which are located on the host cell surface. This binding induces conformational changes in the transmembrane gp41, which facilitates membrane fusion between the viral and host membranes. An understanding of these processes at the molecular level may lead to a direct means of inhibiting HIV infection. As HIV pg41, and the closely related SIV gp41, are heavily glycosylated transmembrane proteins determination of their high resolution structures is a very difficult problem. In an incremental approach, we are studying the structure of (non- glycosylated) functional domains of gp41. The most important region of gp41 is the ectodomain region, located on the outer surface of the viral membrane, which directly mediates membrane fusion events. Both HIV and SIV gp41 ectodomains have been expressed in E.coli. For the SIV gp41, both the NMR and X-ray structures have been solved. Results:High- resolution X-ray (Hyde/Wang) and NMR (Clore/Gronenborn/Caffrey) structures of the SIV gp41 ectodomain have been determined. The structure determined by both methods is a rod-like trimer comprising three parallel N-terminal a-helices assembled as a coiled-coil in the center with three antiparallel C-terminal a-helices packed on the outside with highly flexible loops connecting the inner and outer helices. The loop region connecting the inner and outer helices, although not clearly resolved in the X-ray model, has been modeled in the NMR structure allowing the connectivity’s between inner and outer helices to be established. This region appears responsible for the low solubility of the trimer at physiological pH values resulting in aggregate formation. Our very recent work has led us to postulate these high molecular aggregates of the gp41 are responsible for HIV- associated neurological damage and dementia. Other functional regions of the gp41 protein have been expressed in bacteria, including the cytoplasmic domain, which is though to interact with the HIV matrix protein (a component of the nucleocapsid) as well as various host cellular factors. Significance and future direction:The high-resolution structure determinations of the gp41 ectodomain will allow a more rational approach to the design of novel peptide inhibitors. Based on the physical properties of the gp41, models of membrane fusion have been proposed which involve partially associated protein complexes. These models may also provide useful clues for targeting gp41. A more complete picture of membrane fusion will require more extensive structural work on gp41, especially those regions involved in mediating interaction with gp120. The expression of various engineered gp120 proteins, including the N- and C-terminal domains which are thought to interact with gp41, have been expressed and are being tested.Summary:Envelope proteins on the surface of the human immunodeficiency virus (HIV) that mediate entry of the virus into its host cell have been expressed in bacteria using recombinant DNA methods. The envelope proteins were purified and their structures studied. It is hoped that an understanding of the molecular details of these proteins, vital for the viral life cycle, will aid in the development of reagents and methods for blocking their function. - HIV/SIV, envelope glycoprotein gp41, membrane fusion, recombinant protein expression