Compartmentalization of VP16 in cells infected with recombinant herpes simplex virus expressing VP16-green fluorescent protein fusion proteins
Compartmentalization of VP16 in cells infected with recombinant herpes simplex virus expressing VP16-green fluorescent protein fusion proteins
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DOI：
10.1128/jvi.78.15.8002-8014.2004
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发表时间：
2004-08-01
影响因子：
5.4
通讯作者：
O'Hare, P
中科院分区：
文献类型：
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作者：
La Boissière, S;Izeta, A;O'Hare, P
VP16是单纯疱疹病毒的重要结构蛋白。它在即时-早期转录调控、其他病毒组分的活性调控以及感染性病毒粒子的组装和输出途径中发挥重要作用。为了进一步了解这种多功能蛋白的区隔性，我们构建并表征了表达VP16与绿色荧光蛋白（GFP）连接的重组病毒。这些病毒以几乎正常的动力学和产量进行复制，并将融合蛋白结合到病毒粒子中，产生自荧光颗粒。新合成的VP16-GFP首先在细胞核内以弥漫性模式检测到。核VP16-GFP逐渐被招募到复制区室，这些区室合并成大的球状结构域。感染后10至12小时，可以看到含有VP16-GFP的其他明显病灶，几乎全部位于复制区室的外围。与此同时，在细胞质中观察到明显的积聚，首先以弥漫性模式，然后在囊泡样隔室中积聚，这些隔室以不对称的方式集中，使人想起高尔基体。抑制DNA复制导致细胞核弥漫性分布延长，细胞质积累减少。用布雷菲尔丁治疗破坏了细胞质的水泡模式，导致重新分布的大灶。延时显微镜显示了感染的各种动态特征，包括积极诱导很长的细胞突起（高达100 μ m）。含有VP16的囊泡团在突起中被运输到末端，末端形成球茎末端，并嵌入邻近未感染细胞的膜中。
VP16 is an essential structural protein of herpes simplex virus. It plays important roles in immediate-early transcriptional regulation, in the modulation of the activities of other viral components, and in the pathway of assembly and egress of infectious virions. To gain further insight into the compartmentalization of this multifunctional protein we constructed and characterized recombinant viruses expressing VP16 linked to the green fluorescent protein (GFP). These viruses replicate with virtually normal kinetics and yields and incorporate the fusion protein into the virion, resulting in autofluorescent particles. De novo-synthesized VP16-GFP was first detected in a diffuse pattern within the nucleus. Nuclear VP16-GFP was progressively recruited to replication compartments, which coalesced into large globular domains. By 10 to 12 h after infection additional distinct foci containing VP16-GFP could be seen, almost exclusively located at the periphery of the replication compartments. At the same time pronounced accumulation was observed in the cytoplasm, first in a diffuse pattern and then accumulating in vesicle-like compartments which were concentrated in an asymmetric fashion reminiscent of the Golgi. Inhibition of DNA replication resulted in prolonged diffuse nuclear distribution with minimal cytoplasmic accumulation. Treatment with brefeldin disrupted the cytoplasm vesicular pattern, resulting in redistributed large foci. Time-lapse microscopy demonstrated various dynamic features of infection, including the active induction of very long cellular projections (up to 100 muM). Vesicular clusters containing VP16 were transported within projections to the termini, which developed bulbous ends and appeared to embed into the membranes of adjacent uninfected cells.