Identification of second-site mutations that enhance release and spread of vaccinia virus
Identification of second-site mutations that enhance release and spread of vaccinia virus
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DOI：
10.1128/jvi.76.22.11637-11644.2002
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发表时间：
2002-11-01
影响因子：
5.4
通讯作者：
Moss, B
中科院分区：
文献类型：
--
作者：
Katz, E;Wolffe, E;Moss, B
大多数痘苗病毒株在细胞单层中的传播主要通过细胞外被包裹的病毒粒子发生，这些病毒粒子附着在含有肌动蛋白的微绒毛的尖端，其次是通过释放的病毒粒子的扩散。病毒粒子附着在细胞表面的机制尚不清楚，尽管可能涉及几种病毒蛋白。目前的研究是在以下前提下开始的：增加病毒释放的自发突变将通过繁殖无法通过肌动蛋白尾巴传播的病毒来自然选择。从一个形成小而圆的空斑的A36R缺失突变体开始，分离到5个由于彗星或卫星空斑的形成而增强传播的独立病毒克隆。对分离株的病毒膜糖蛋白基因进行了测序，其中4株有突变，导致A33R蛋白的C端截断，1株用丝氨酸取代了13511蛋白中的第189条。彗星形成表型分别通过含有突变序列或自然序列的DNA通过同源重组而特异性地复制或逆转。第二位突变体比亲本A36R缺失突变体释放的胞外被膜病毒多得多，这解释了它们在组织培养中的选择以及它们的彗星形成表型。这些数据表明，13511蛋白和A33R蛋白的C-末端区域参与了与细胞相关的包膜病毒粒子与细胞的黏附。尽管它们在培养细胞中具有选择性优势，但当通过鼻腔途径给小鼠注射A36R缺失突变体时，它们的毒性并不比A36R缺失突变体好得多。
The spread of most strains of vaccinia virus in cell monolayers occurs predominantly via extracellular enveloped virions that adhere to the tips of actin-containing microvilli and to a lesser extent via diffusion of released virions. The mechanism by which virions adhere to the cell surface is unknown, although several viral proteins may be involved. The present investigation was initiated with the following premise: spontaneous mutations that increase virus release will be naturally selected by propagating a virus unable to spread by means of actin tails. Starting with an A36R deletion mutant that forms small, round plaques, five independent virus clones with enhanced spread due to the formation of comet or satellite plaques were isolated. The viral membrane glycoprotein genes of the isolates were sequenced; four had mutations causing C-terminal truncations of the A33R protein, and one had a serine replacing proline 189 of the 13511 protein. The comet-forming phenotype was specifically reproduced or reversed by homologous recombination using DNA containing the mutated or natural sequence, respectively. Considerably more extracellular enveloped virus was released into the medium by the second-site mutants than by the parental A36R deletion mutant, explaining their selection in tissue culture as well as their comet-forming phenotype. The data suggest that the 13511 protein and the C-terminal region of the A33R protein are involved in adherence of cell-associated enveloped virions to cells. In spite of their selective advantage in cultured cells, the second-site mutants were not delectably more virulent than the A36R deletion mutant when administered to mice by the intranasal route.