Gallego-Gómez Juan Carlos, Risco Cristina, Rodríguez Dolores, Cabezas Pilar, Guerra Susana, Carrascosa José L, Esteban Mariano
Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus Universidad Autónoma, 28049 Madrid, Spain.
J Virol. 2003 Oct;77(19):10606-22. doi: 10.1128/jvi.77.19.10606-10622.2003.
Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.
基于减毒修饰安卡拉痘苗病毒（MVA）的活重组体是针对多种疾病和肿瘤的潜在疫苗候选物。为了更好地了解MVA作为人类疫苗的功效，我们通过共聚焦显微镜和电子显微镜方法分析了MVA在感染人HeLa细胞过程中诱导的形态变化和形态发生阶段，并与其他痘苗病毒（VV）株进行了比较：野生型西储（WR）株、安卡拉株和纽约市卫生局（NYCBH）株。共聚焦显微镜研究表明，MVA感染会改变细胞骨架，产生细长细胞（双极），且不会形成特征性的肌动蛋白尾。在连接相邻细胞的突起中几乎检测不到病毒粒子。相比之下，感染WR株、安卡拉株和NYCBH株的细胞呈现出星状（多极）或圆形形态，并带有肌动蛋白尾。对感染MVA的HeLa细胞进行的详细透射电子显微镜分析显示，与感染其他VV株的细胞相比，其精细超微结构和病毒中间体数量存在重要差异。在感染MVA的HeLa细胞中，最丰富的病毒形式是细胞内未成熟病毒，很少有中间体达到细胞内成熟病毒（IMV）形式，且处于不同成熟阶段，这些IMV比感染其他VV株的细胞中的IMV形状更圆。来自感染MVA细胞的“IMV”具有异常的内部结构（“非典型”病毒），核心-包膜相互作用可能发生改变，并且无法显著获得额外的双层包膜以形成细胞内包膜病毒。潜在的细胞相关包膜病毒非常稀少。我们的研究结果表明，MVA在人类细胞中会促使细胞发生特征性形态变化，并能够达到IMV阶段，但这些病毒粒子结构不正常，形态发生途径的后续步骤被阻断。
