Interaction study of rice stripe virus proteins reveals a region of the nucleocapsid protein (NP) required for NP self-interaction and nuclear localization.
Rice stripe virus (RSV), which belongs to the genus Tenuivirus, is an emergent virus problem. The RSV genome is composed of four single-strand RNAs (RNA1-RNA4) and encodes seven proteins. We investigated interactions between six of the RSV proteins by yeast-two hybrid (Y2H) assay in vitro and by bimolecular fluorescence complementation (BiFC) in planta. Y2H identified self-interaction of the nucleocapsid protein (NP) and NS3, while BiFC revealed self-interaction of NP, NS3, and NCP. To identify regions(s) and/or crucial amino acid (aa) residues required for NP self-interaction, we generated various truncated and aa substitution mutants. Y2H assay showed that the N-terminal region of NP (aa 1-56) is necessary for NP self-interaction. Further analysis with substitution mutants demonstrated that additional aa residues located at 42-47 affected their interaction with full-length NP. These results indicate that the N-terminal region (aa 1-36 and 42-47) is required for NP self-interaction. BiFC and co-localization studies showed that the region required for NP self-interaction is also required for NP localization at the nucleus. Overall, our results indicate that the N-terminal region (aa 1-47) of the NP is important for NP self-interaction and that six aa residues (42-47) are essential for both NP self-interaction and nuclear localization.
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Sen
S
Lian
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Won Kyong
WK
Cho
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Yeonhwa
Y
Jo
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea.
Sang-Min
SM
Kim
Department of Agricultural Biotechnology and Plant Genomics and Breeding Institute, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Electronic address: kookkim@snu.ac.kr.
Kook-Hyung
KH
Kim
D004252:DNA Mutational Analysis; D019913:Nuclear Localization Signals; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D029364:Tenuivirus / Q000235:genetics* / Q000502:physiology; D020798:Two-Hybrid System Techniques
D019913:Nuclear Localization Signals; D019590:Nucleocapsid Proteins
Nuclear localization; Nucleocapsid protein; Protein–protein interactions; Rice stripe virus; Yeast two-hybrid system