Carter Gemma C, Rodger Gaener, Murphy Brendan J, Law Mansun, Krauss Oliver, Hollinshead Michael, Smith Geoffrey L
Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK.
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
J Gen Virol. 2003 Sep;84(Pt 9):2443-2458. doi: 10.1099/vir.0.19271-0.
Infection with Vaccinia virus (VV) produces several distinct virions called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). In this report, we have investigated how incoming virus cores derived from IMV are transported within the cell. To do this, recombinant VVs (vA5L-EGFP-N and vA5L-EGFP-C) were generated in which the A5L virus core protein was fused with the enhanced green fluorescent protein (EGFP) at the N or C terminus. These viruses were viable, induced formation of actin tails and had a plaque size similar to wild-type. Immunoblotting showed the A5L-EGFP fusion protein was present in IMV particles and immunoelectron microscopy showed that the fusion protein was incorporated into VV cores. IMV made by vA5L-EGFP-N were used to follow the location and movement of cores after infection of PtK(2) cells. Confocal microscopy showed that virus cores were stained with anti-core antibody only after they had entered the cell and, once intracellular, were negative for the IMV surface protein D8L. These cores co-localized with microtubules and moved in a stop-start manner with an average speed of 51.8 (+/-3.9) microm min(-1), consistent with microtubular movement. Treatment of cells with nocodazole or colchicine inhibited core movement, but addition of cytochalasin D did not. These data show that VV cores derived from IMV use microtubules for intracellular transport after entry.
痘苗病毒（VV）感染会产生几种不同的病毒粒子，称为细胞内成熟病毒（IMV）、细胞内包膜病毒（IEV）、细胞相关包膜病毒（CEV）和细胞外包膜病毒（EEV）。在本报告中，我们研究了源自IMV的传入病毒核心在细胞内是如何运输的。为此，构建了重组痘苗病毒（vA5L-EGFP-N和vA5L-EGFP-C），其中A5L病毒核心蛋白在N端或C端与增强型绿色荧光蛋白（EGFP）融合。这些病毒具有活性，能诱导肌动蛋白尾的形成，且噬斑大小与野生型相似。免疫印迹显示A5L-EGFP融合蛋白存在于IMV颗粒中，免疫电子显微镜显示该融合蛋白被整合到痘苗病毒核心中。用vA5L-EGFP-N产生的IMV用于追踪感染PtK(2)细胞后核心的位置和移动。共聚焦显微镜显示，病毒核心只有在进入细胞后才会被抗核心抗体染色，并且一旦进入细胞内，IMV表面蛋白D8L呈阴性。这些核心与微管共定位，并以停止-启动的方式移动，平均速度为51.8（±3.9）μm/min，这与微管运动一致。用诺考达唑或秋水仙碱处理细胞会抑制核心运动，但加入细胞松弛素D则不会。这些数据表明，源自IMV的痘苗病毒核心在进入细胞后利用微管进行细胞内运输。
