Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly
Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly
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DOI：
10.1128/jvi.01371-15
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发表时间：
2015-09
影响因子：
5.4
通讯作者：
Timothy K. Soh;S. Whelan
中科院分区：
文献类型：
--
作者：
Timothy K. Soh;S. Whelan
摘要水泡性口炎病毒(VSV)的组装需要病毒核糖核蛋白(RNP)核心与基质蛋白(M)在从质膜萌发过程中缩合。核糖核蛋白的核心是完全被核衣壳蛋白(N)包裹的负义基因组RNA，并被一个磷酸蛋白(P)和大聚合酶蛋白(L)结合。为了研究单个病毒颗粒的组装，我们用荧光蛋白标记了M和P。我们从插入了M基因的病毒库中选择了一种含有荧光M的复制能力强的病毒，并将其与我们之前描述的含有荧光P病毒的病毒颗粒相结合。含有这些融合的病毒颗粒保持了与野生型VSV相同的子弹形状，但颗粒长度略有增加，反映了基因组大小的增加。对释放的颗粒的成像显示，M和P组装到病毒粒子中的数量发生了变化，这与柔性包装机制一致。我们使用重组体进一步研究了M中晚期结构域的重要性，这些结构域在萌发过程中用于招募运输所需的内体分选复合体(ESCRT)机械。晚期结构域的突变导致病毒粒子的积累，这些病毒粒子无法从质膜上剥离。用标记了增强绿色荧光蛋白的M和标记了mCherry变异体的M混合感染细胞释放的单个病毒粒子的成像显示，对晚期区域突变被突变失活的病毒粒子有强烈的偏见。相反，两个变异体的细胞内表达和膜结合没有改变。这些研究为成像颗粒组装提供了新的工具，并提高了我们现有的VSV组装模型的分辨率。水疱性口炎病毒(VSV)颗粒的重要组装要求病毒复制机制、基质蛋白(M)和糖蛋白分别运输到质膜。基质蛋白含有一个被称为“晚期结构域”的基序，该基序与宿主转运所需的内体分选复合体(ESCRT)机制相结合，以促进病毒颗粒的释放。通过突变使晚期结构域失活，导致在释放点停止的病毒粒子积累。在这里描述的研究中，我们开发了新的工具来研究VSV组装，方法是将荧光蛋白融合到M和复制机制的一个组成部分--磷蛋白(P)上。我们使用这些工具来证明M的晚期结构域是有效地整合到病毒颗粒中所必需的，并且这些颗粒含有不同数量的M和P。
ABSTRACT Vesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV. IMPORTANCE Assembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.