Cytoskeletal proteins inside human immunodeficiency virus type 1 virions
Cytoskeletal proteins inside human immunodeficiency virus type 1 virions
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DOI：
10.1128/jvi.70.11.7734-7743.1996
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发表时间：
1996-11-01
影响因子：
5.4
通讯作者：
Henderson, LE
中科院分区：
文献类型：
--
作者：
Ott, DE;Coren, LV;Henderson, LE
我们通过分析枯草杆菌毒素消化的颗粒，在人类免疫缺陷病毒1型(HTV-1)病毒粒子中鉴定了三种类型的细胞骨架蛋白。用酶消化HIV-1病毒粒子，蔗糖密度离心法分离处理后的病毒粒子。这种方法可以去除外部病毒蛋白和与污染病毒粒子制剂的微泡相关的蛋白。由于病毒粒子内的蛋白质受到病毒脂膜的保护，使其不被消化，因此可以在治疗后分离和分析它们。这里介绍的实验表明，这个过程去除了超过95%的与微泡相关的蛋白质。用高压液相色谱、蛋白质测序和免疫印迹分析了H9和CEM(Ss)细胞株消化的HIV-1(MN)颗粒中的蛋白质。数据显示，与Gag的摩尔水平相比，病毒粒子中存在三种不同浓度的细胞骨架蛋白：肌动蛋白(约10%至15%)、Ezrin和moesin(约2%)以及Cofilin(约2%至10%)。我们对病毒颗粒内蛋白质的分析发现，CW-平滑肌肌动蛋白和Moesin的蛋白水解性片段在可能被HIV-1蛋白酶识别的位置被切割。这些裂解产物不存在于未感染细胞的微泡中。因此，这些加工过的蛋白质很可能是由HIV-1蛋白酶消化产生的。这些片段的存在，以及少数特定的细胞骨架蛋白被掺入病毒粒子中，表明细胞骨架蛋白和病毒蛋白之间存在活跃的相互作用。
We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HTV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles mere isolated by sucrose density centrifugation. This method removes both exterior viral proteins and proteins associated with microvesicles that contaminate virion preparations. Since the proteins inside the virion are protected from digestion by the viral lipid envelope, they can be isolated and analyzed after treatment. Experiments presented here demonstrated that this procedure removed more than 95% of the protein associated with microvesicles. Proteins in digested HIV-1(MN) particles from infected H9 and CEM(ss) cell lines were analyzed by high-pressure liquid chromatography, protein sequencing, and immunoblotting. The data revealed that three types of cytoskeletal proteins are present in virions at different concentrations relative to the molar level of Gag: actin (approximately 10 to 15%), ezrin and moesin (approximately 2%), and cofilin (approximately 2 to 10%). Our analysis of proteins within virus particles detected proteolytic fragments of cw-smooth muscle actin and moesin that were cleaved at sites which might be recognized by HIV-1 protease. These cleavage products are not present in microvesicles from uninfected cells. Therefore, these processed proteins are most probably produced by HIV-1 protease digestion. The presence of these fragments, as well as the incorporation of a few specific cytoskeletal proteins into virions, suggests an active interaction between cytoskeletal and, viral proteins.