title: Critical regions and residues for self-interaction of grapevine leafroll-associated virus 2 protein p24.
abstract: The 24-kDa protein (p24) encoded by grapevine leafroll-associated virus 2 (GLRaV-2) is an RNA-silencing suppressor. In this work, a yeast two-hybrid system (YTHS) and bimolecular fluorescence complementation analyses showed that GLRaV-2 p24 can interact with itself, and that this interaction occurs in the cytoplasm of Nicotiana benthamiana cells. To identify the functional region(s) and crucial amino acid residues required for p24 self-interaction, various truncated and substitution mutants were generated. YTHS assay showed that in both homologous pairing and pairing with the wild-type p24, the functional regions mapped to aa 10-180 or 1-170 which contain, respectively, all seven α-helices or the first six α-helices and the N-terminal end (aa 1-9) of the protein. When only the full-length p24 was an interaction partner, the functional region of aa 1-170 could be further mapped to aa 1-140 which contains four α-helices plus most of the fifth α-helix. Further analysis with substitution mutants demonstrated that hydrophobic residues I35/F38/V85/V89/W149 and V162/L169/L170, which may, respectively, mediate the inter-domain interaction of the same p24 monomer and the tail-to-tail association between two p24 counterparts, are crucial for homotypic p24-p24 interaction. In addition, substitution of two basic residues-R2 or R86-of p24, which may play important functional roles in RNA binding, did not seem to affect self-interaction of the mutants in yeast but had obvious effects in plant cells. Taken together, our results demonstrate the functional regions and crucial amino acids for p24 self-interaction.
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.
authors.forename: Ran
authors.initials: R
authors.lastname: Guo
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.
authors.forename: Mingjun
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.
authors.forename: Ming
authors.initials: M
authors.lastname: Feng
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China.
authors.forename: Xianyou
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Plant Pathology, China Agricultural University, Beijing 100193, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Pomology/Lab of Stress Physiology and Molecular Biology for Tree Fruits, A Key Lab of Beijing Municipality, China Agricultural University, Beijing 100193, China. Electronic address: chengyuqin@cau.edu.cn.
authors.forename: Yuqin
authors.initials: Y
authors.lastname: Cheng
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017790:Closterovirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000821:virology; D015870:Gene Expression; D016153:Genes, Suppressor; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009154:Mutation; D059828:Plant Cells / Q000821:virology*; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D054730:Protein Interaction Domains and Motifs; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000821:virology; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins
keywords: Bimolecular fluorescence complementation; GLRaV-2; RNA-silencing suppressor; Self-interaction; Yeast two-hybrid system; p24 protein