Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture
Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture
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甜菜黑焦病病毒感染期间内质网膜内陷囊泡的形态发生：辅助复制蛋白的作用和三维结构的新意义
DOI：
10.1128/jvi.00401-15
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发表时间：
2015-06-01
影响因子：
5.4
通讯作者：
Zhang, Yongliang
中科院分区：
文献类型：
--
作者：
Cao, Xiuling;Jin, Xuejiao;Zhang, Yongliang
摘要所有特征良好的正链RNA病毒[(+)RNA病毒]都能诱导宿主膜结合的病毒复制复合体(VRC)的形成，但VRC形成的潜在机制和机制仍不清楚。本文报道了甜菜黑焦病病毒(BBSV)复制复合体的生物发生和拓扑结构。在感染BBSV的本氏烟草细胞中，观察到典型的内质网(ER)聚集和囊泡的典型细胞病理变化。辅助复制蛋白p23和双链RNA(DsRNA)的免疫金标记法显示，内质网来源的膜球为BBSV的复制提供了场所。进一步的研究表明，p23在内质网重排中起着至关重要的作用。三维电子断层扫描显示形成多个内质网起源的囊泡包。每个囊泡包裹着几个到数百个独立的球体，这些球体是内质网膜内陷到管腔中的。引人注目的是，这些囊泡通过小管相互连接，这种重排事件在其他病毒诱导的膜重组中是罕见的。电子断层扫描还重建了球体内的纤维内容物，显示了不同的结构。据我们所知，我们的结果首次对植物(+)RNA病毒膜结合的VRCs进行了三维超微结构分析，这将有助于从机制上更好地理解植物(+)RNA病毒复制复合体的组织和微环境。病毒复制复合体对所有已知正链RNA病毒的重要性组装依赖于宿主细胞内膜的广泛重塑。甜菜黑焦病病毒是番茄病毒科的一种坏死病毒，它侵入内质网(ER)膜，在感染细胞中形成球状。病毒复制中间体双链RNA和病毒辅助复制蛋白p23都定位于这种病毒小球中，表明这些都是产生后代病毒RNA的位点。此外，BBSV p23蛋白在本氏新月球藻中瞬时表达时，可以在一定程度上重组内质网。电子断层扫描分析解决了这种球体的三维(3D)结构，这些球体通过颈状结构连接到细胞质。值得注意的是，不同数量的球体被包裹在内质网起源的囊泡包中，这些囊泡通过管状结构相互连接。我们的结果对于进一步理解正链RNA病毒复制的机制具有重要意义。
ABSTRACT All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes. IMPORTANCE Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus, a necrovirus in the family Tombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.