Rice black streaked dwarf virus P9-1, an alpha-helical protein, self-interacts and forms viroplasms in vivo.
Replication and assembly of viruses from the family Reoviridae are thought to take place in discrete cytoplasmic inclusion bodies, commonly called viral factories or viroplasms. Rice black streaked dwarf virus (RBSDV) P9-1, a non-structural protein, has been confirmed to accumulate in these intracellular viroplasms in infected plants and insects. However, little is known about its exact function. In this study, P9-1 of RBSDV-Baoding was expressed in Escherichia coli as a His-tagged fusion protein and analysed using biochemical and biophysical techniques. Mass spectrometry and circular dichroism spectroscopy studies showed that P9-1 was a thermostable, alpha-helical protein with a molecular mass of 41.804 kDa. A combination of gel-filtration chromatography, chemical cross-linking and a yeast two-hybrid assay was used to demonstrate that P9-1 had the intrinsic ability to self-interact and form homodimers in vitro and in vivo. Furthermore, when transiently expressed in Arabidopsis protoplasts, P9-1 formed large, discrete viroplasm-like structures in the absence of infection or other RBSDV proteins. Taken together, these results suggest that P9-1 is the minimal viral component required for viroplasm formation and that it plays an important role in the early stages of the virus life cycle by forming intracellular viroplasms that serve as the sites of virus replication and assembly.
Zhang
Chaozheng
C
Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Liu
Yueyong
Y
California Pacific Medical Center Research Institute, San Francisco, CA 94107, USA.
Liu
Liyue
L
Bureau of Animal Husbandry and Fishery of Xiangxiang, Xiangxiang 411400, PR China.
Lou
Zhiyong
Z
Laboratory of Structural Biology, School of Life Sciences and Bio-Engineering, Tsinghua University, Beijing 100084, PR China.
Zhang
Hongyan
H
Laboratory of Structural Biology, School of Life Sciences and Bio-Engineering, Tsinghua University, Beijing 100084, PR China.
Miao
Hongqin
H
Hebei Academy of Agricultural Sciences, Baoding 071000, PR China.
Hu
Xuebo
X
Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Pang
Yanping
Y
Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Qiu
Bingsheng
B
Center for Agricultural Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
D017360:Arabidopsis / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D007181:Inclusion Bodies, Viral / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012087:Reoviridae / Q000502:physiology*; D017422:Sequence Analysis, DNA; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins