Ott D E, Coren L V, Kane B P, Busch L K, Johnson D G, Sowder R C, Chertova E N, Arthur L O, Henderson L E
AIDS Vaccine Program, National Cancer Institute, Frederick Cancer Research and Development Center, Maryland 21702-1201, USA.
J Virol. 1996 Nov;70(11):7734-43. doi: 10.1128/JVI.70.11.7734-7743.1996.
We have identified three types of cytoskeletal proteins inside human immunodeficiency virus type 1 (HIV-1) virions by analyzing subtilisin-digested particles. HIV-1 virions were digested with protease, and the treated particles were 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 alpha-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.
通过分析枯草杆菌蛋白酶消化后的颗粒，我们在1型人类免疫缺陷病毒（HIV-1）病毒粒子内鉴定出了三种细胞骨架蛋白。用蛋白酶消化HIV-1病毒粒子，然后通过蔗糖密度离心法分离处理后的颗粒。该方法可去除外部病毒蛋白以及与污染病毒粒子制剂的微泡相关的蛋白。由于病毒粒子内部的蛋白受到病毒脂质包膜的保护而免受消化，因此在处理后可以对其进行分离和分析。此处呈现的实验表明，该程序去除了超过95%与微泡相关的蛋白。通过高压液相色谱、蛋白质测序和免疫印迹分析了来自感染的H9和CEM(ss)细胞系的经消化的HIV-1（MN）颗粒中的蛋白质。数据显示，相对于Gag的摩尔水平，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%）、埃兹蛋白和膜突蛋白（约2%）以及丝切蛋白（约2%至10%）。我们对病毒颗粒内蛋白质的分析检测到α-平滑肌肌动蛋白和膜突蛋白的蛋白水解片段，这些片段在可能被HIV-1蛋白酶识别的位点被切割。这些切割产物不存在于未感染细胞的微泡中。因此，这些加工后的蛋白很可能是由HIV-1蛋白酶消化产生的。这些片段的存在，以及少数特定细胞骨架蛋白掺入病毒粒子，表明细胞骨架蛋白和病毒蛋白之间存在活跃的相互作用。
