Newcomb W W, Homa F L, Thomsen D R, Trus B L, Cheng N, Steven A, Booy F, Brown J C
Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
J Virol. 1999 May;73(5):4239-50. doi: 10.1128/JVI.73.5.4239-4250.1999.
An in vitro system is described for the assembly of herpes simplex virus type 1 (HSV-1) procapsids beginning with three purified components, the major capsid protein (VP5), the triplexes (VP19C plus VP23), and a hybrid scaffolding protein. Each component was purified from insect cells expressing the relevant protein(s) from an appropriate recombinant baculovirus vector. Procapsids formed when the three purified components were mixed and incubated for 1 h at 37 degrees C. Procapsids assembled in this way were found to be similar in morphology and in protein composition to procapsids formed in vitro from cell extracts containing HSV-1 proteins. When scaffolding and triplex proteins were present in excess in the purified system, greater than 80% of the major capsid protein was incorporated into procapsids. Sucrose density gradient ultracentrifugation studies were carried out to examine the oligomeric state of the purified assembly components. These analyses showed that (i) VP5 migrated as a monomer at all of the protein concentrations tested (0.1 to 1 mg/ml), (ii) VP19C and VP23 migrated together as a complex with the same heterotrimeric composition (VP19C1-VP232) as virus triplexes, and (iii) the scaffolding protein migrated as a heterogeneous mixture of oligomers (in the range of monomers to approximately 30-mers) whose composition was strongly influenced by protein concentration. Similar sucrose gradient analyses performed with mixtures of VP5 and the scaffolding protein demonstrated the presence of complexes of the two having molecular weights in the range of 200,000 to 600,000. The complexes were interpreted to contain one or two VP5 molecules and up to six scaffolding protein molecules. The results suggest that procapsid assembly may proceed by addition of the latter complexes to regions of growing procapsid shell. They indicate further that procapsids can be formed in vitro from virus-encoded proteins only without any requirement for cell proteins.
本文描述了一种体外系统，该系统可从三种纯化成分开始组装单纯疱疹病毒1型（HSV-1）原衣壳，这三种成分分别是主要衣壳蛋白（VP5）、三聚体（VP19C加VP23）和一种杂合支架蛋白。每种成分均从表达相应蛋白质的昆虫细胞中纯化而来，这些昆虫细胞由合适的重组杆状病毒载体转染。将三种纯化成分混合，并在37℃孵育1小时后可形成原衣壳。发现以这种方式组装的原衣壳在形态和蛋白质组成上与从含有HSV-1蛋白质的细胞提取物体外形成的原衣壳相似。当纯化系统中支架蛋白和三聚体蛋白过量时，超过80%的主要衣壳蛋白会掺入原衣壳中。进行了蔗糖密度梯度超速离心研究，以检查纯化的组装成分的寡聚状态。这些分析表明：（i）在所有测试的蛋白质浓度（0.1至1mg/ml）下，VP5均以单体形式迁移；（ii）VP19C和VP23作为复合物一起迁移，其异源三聚体组成（VP19C1-VP232）与病毒三聚体相同；（iii）支架蛋白以寡聚体的异质混合物形式迁移（单体至约30聚体范围内），其组成受蛋白质浓度的强烈影响。对VP5和支架蛋白混合物进行的类似蔗糖梯度分析表明，两者形成了分子量在200,000至600,000范围内的复合物。这些复合物被认为包含一个或两个VP5分子以及多达六个支架蛋白分子。结果表明，原衣壳组装可能是通过将后一种复合物添加到正在生长的原衣壳壳区域来进行的。它们进一步表明，仅从病毒编码的蛋白质就可以在体外形成原衣壳，而无需任何细胞蛋白质。
