Interaction of Mal de Río Cuarto virus (Fijivirus genus) proteins and identification of putative factors determining viroplasm formation and decay

• Published in: Virus research Vol. 230; pp. 19 - 28
• Main Authors: Llauger, Gabriela, de Haro, Luis Alejandro, Alfonso, Victoria, del Vas, Mariana
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.02.2017
• Subjects: Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - metabolism; agroinfiltration; Amino Acid Motifs; Amino Acid Sequence; Conserved Sequence; Fijivirus; fluorescence; Gene Expression; glutamic acid; Host-Pathogen Interactions; Inclusion Bodies - chemistry; Inclusion Bodies - metabolism; Inclusion Bodies - virology; leaves; membrane proteins; MRCV; Nicotiana - virology; Nicotiana benthamiana; Oryza - virology; PEST sequence; Plant Diseases - virology; Plant Leaves - metabolism; Plant Leaves - ultrastructure; Plant Leaves - virology; proline; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Proteolysis; protoplasts; Protoplasts - metabolism; Protoplasts - ultrastructure; Protoplasts - virology; Reoviridae; Reoviridae - genetics; Reoviridae - metabolism; rice; Sequence Alignment; Sequence Homology, Amino Acid; serine; threonine; Two-Hybrid System Techniques; viral inclusion bodies; viral nonstructural proteins; Viral Nonstructural Proteins - chemistry; Viral Nonstructural Proteins - genetics; Viral Nonstructural Proteins - metabolism; viroplasm; viruses; Y2H; PEST sequence; MRCV; viroplasm; Reoviridae; Y2H
• ISSN: 0168-1702; 1872-7492; 1872-7492
• DOI: 10.1016/j.virusres.2017.01.002

•MRCV P6 self-interacts and interacts with the major viroplasm protein P9-1.•P6 residues 450–788 are necessary for P6/P6 and P6/P9-1 interactions.•P9-1 contains a C-terminal arm that affects its dimerization.•P6 and P9-1 contain PEST sequences that might affect their degradation. Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function. Interactions involving P6 and P9-1 were confirmed by bimolecular fluorescence complementation (BiFC) in rice protoplasts. We demonstrated that a region including a predicted coiled-coil domain within the C-terminal moiety of P6 was necessary for P6/P6 and P6/P9-1 interactions. In turn, a short C-terminal arm was necessary for the previously reported P9-1 self-interaction. Transient expression of these proteins by agroinfiltration of Nicotiana benthamiana leaves showed very low accumulation levels and further in silico analyses allowed us to identify conserved PEST degradation sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] within P6 and P9-1. The removal of these PEST sequences resulted in a significant increase of the accumulation of both proteins.