Multiple interactions among proteins encoded by the mite-transmitted wheatstreak mosaic tritimovirus

The genome organization of the mite-transmitted wheat streak mosaic virus ( WSMV) appears to parallel that of members of the Potyviridae with monoparti te genomes, but there are substantial amino acid dissimilarities with other potyviral polyproteins. To initiate studies on the functions of WSMV-encod ed proteins, a protein interaction map was generated using a yeast two-hybr id system. Because the pathway of proteolytic maturation of the WSMV polypr otein has not been experimentally determined, random libraries of WSMV cDNA were made both in DNA-binding domain and activation domain plasmid vectors and introduced into yeast. Sequence analysis of multiple interacting pairs revealed that interactions largely occurred between domains within two gro ups of proteins. The first involved interactions among nuclear inclusion pr otein a, nuclear inclusion protein b, and coat protein (CP), and the second involved helper component-proteinase (HC-Pro) and cylindrical inclusion pr otein (CI). Further immunoblot and deletion mapping analyses of the interac tions suggest that subdomains of CI, HC-Pro, and P1 interact with one anoth er. The two-hybrid assay was then performed using full-length genes of CI, HC-Pro, P1, P3, and CP, but no heterologous interactions were detected. In vitro binding assay using glutathione-S-transferase fusion proteins and in vitro translation products, however, revealed mutual interactions among Cl, HC-Pro, P1, and P3. The failure to detect interactions between full-length proteins by the two-hybrid assay might be due to adverse effects of expres sion of viral proteins in yeast cells. The capacity to participate in multi ple homomeric and heteromeric molecular interactions is consistent with the pleiotropic nature of many potyviral gene mutants and suggests mechanisms for regulation of various viral processes via a network of viral protein co mplexes. (C) 2000 Academic Press.