Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-Tagged Gc Glycoprotein
Visualizing the Replication Cycle of Bunyamwera Orthobunyavirus Expressing Fluorescent Protein-Tagged Gc Glycoprotein
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DOI：
10.1128/jvi.00902-10
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发表时间：
2010-09-01
影响因子：
5.4
通讯作者：
Elliott, Richard M.
中科院分区：
文献类型：
--
作者：
Shi, Xiaohong;van Mierlo, Joel T.;Elliott, Richard M.
布尼亚韦拉病毒（Bunyamwera virus，BUNV）是布尼亚病毒科（Bunyaviridae）的原型，也是正布尼亚病毒属（Orthobunyavirus）的原型，其病毒粒子糖蛋白Gn和Gc由中等（M）RNA基因组片段编码，并且参与病毒的附着和进入。合成后，Gn和Gc在内质网（ER）中形成异源二聚体，并转运到高尔基体区室进行病毒组装。先前显示Gc胞外域的N-末端一半在细胞培养物中对于病毒复制是不稳定的（X. Shi，J. Goli，G.克拉克，K. Brauburger和R. M.埃利奥特，J.将军。90：2483-2492，2009）。在这项研究中，荧光蛋白的编码序列，无论是增强型绿色荧光蛋白（eGFP）或mCherry荧光蛋白，融合到截短的Gc的N端，和两个重组BUNV（rBUNGc-eGFP和rBUNGc-mCherry）通过反向遗传学拯救。重组病毒在紫外光下显示明亮的自发荧光，并能在各种哺乳动物细胞系中复制。rBUNGc-mCherry在10代内完全稳定，而在rBUNGc-eGFP的嵌合Gc-eGFP蛋白中发生内部框内缺失，导致第5代和第7代之间的荧光损失。重组病毒的自发荧光允许可视化感染周期的不同阶段，包括病毒附着到细胞表面，病毒颗粒在高尔基体膜中出芽，以及病毒诱导的高尔基体区室在感染后期的形态变化。荧光蛋白标记的病毒将是活细胞成像研究的有价值的试剂，用于真实的研究病毒进入、出芽和形态发生。
The virion glycoproteins Gn and Gc of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family and also of the Orthobunyavirus genus, are encoded by the medium (M) RNA genome segment and are involved in both viral attachment and entry. After their synthesis Gn and Gc form a heterodimer in the endoplasmic reticulum (ER) and transit to the Golgi compartment for virus assembly. The N-terminal half of the Gc ectodomain was previously shown to be dispensable for virus replication in cell culture (X. Shi, J. Goli, G. Clark, K. Brauburger, and R. M. Elliott, J. Gen. Virol. 90: 2483-2492, 2009.). In this study, the coding sequence for a fluorescent protein, either enhanced green fluorescent protein (eGFP) or mCherry fluorescent protein, was fused to the N terminus of truncated Gc, and two recombinant BUNVs (rBUNGc-eGFP and rBUNGc-mCherry) were rescued by reverse genetics. The recombinant viruses showed bright autofluorescence under UV light and were competent for replication in various mammalian cell lines. rBUNGc-mCherry was completely stable over 10 passages, whereas internal, in-frame deletions occurred in the chimeric Gc-eGFP protein of rBUNGc-eGFP, resulting in loss of fluorescence between passages 5 and 7. Autofluorescence of the recombinant viruses allowed visualization of different stages of the infection cycle, including virus attachment to the cell surface, budding of virus particles in Golgi membranes, and virus-induced morphological changes to the Golgi compartment at later stages of infection. The fluorescent protein-tagged viruses will be valuable reagents for live-cell imaging studies to investigate virus entry, budding, and morphogenesis in real time.