title: 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.
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.
  
  
  OBJECTIVE
  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.
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Jin
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Branch of China National Center for Protein Sciences, Tsinghua University, Beijing, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Institute of Biotechnology, Zhejiang University, Hangzhou, People's Republic of China.
authors.forename: Chunyan
authors.initials: C
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Xianbing
authors.initials: X
authors.lastname: Wang
authors.affiliation: Institute of Biotechnology, Zhejiang University, Hangzhou, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Hong
authors.affiliation: Department of Plant Pathology, Physiology, and Weed Science, Virginia Tech University, Blacksburg, Virginia, USA.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Dawei
authors.initials: D
authors.lastname: Li
authors.affiliation: State Key Laboratory of Agro-Biotechnology and Ministry of Agriculture Key Laboratory of Soil Microbiology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China cauzhangyl@cau.edu.cn.
authors.forename: Yongliang
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D055032:Electron Microscope Tomography; D004721:Endoplasmic Reticulum / Q000648:ultrastructure / Q000821:virology*; D054884:Host-Pathogen Interactions; D016253:Microscopy, Immunoelectron; D014026:Nicotiana; D019183:Tombusviridae / Q000502:physiology* / Q000648:ultrastructure; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D014764:Viral Proteins
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