Importance of the C-terminal domain of soybean mosaic virus coat protein for subunit interactions

Abstract

The potyvirus coat protein (CP) is involved in aphid transmission, cell-to-cell movement and virus assembly, not only by binding to viral RNA, but also by self-interaction or interactions with other factors. In this study, a number of CP mutants of Soybean mosaic virus (SMV) containing deletions and site-directed mutations were generated and cloned into yeast two-hybrid vectors. Interaction was confirmed by the expression of reporter genes, including HIS3, ADE2 and MEL1, in yeast strain AH109. Deletion of the C-terminal region of the CP caused loss of the CP-CP self-interaction ability detected in CP mutants with the C-terminal region. Alanine substitution at the amino acid positions R190, E191, E212, R245, H246 and R249 disrupted CP-CP interaction, whereas substitutions at the amino acid positions R188, D189, D198, K205, K218 and D250 did not. These results indicate that the C-terminal region of SMV CP may contain a domain(s) or amino acids required for CP-CP interaction and virus assembly.Diseases caused by Soybean mosaic virus (SMV) have been reported in many countries and have significantly reduced crop yields and quality worldwide (Jayaram et al., 1992;Hill, 1999). Despite the economic importance of SMV, relatively little is understood about the detailed molecular interactions between viral proteins and the host proteins that occur during viral infections. The viral RNA-dependent RNA polymerase (nuclear inclusion protein b) of Tobacco vein mottling virus reportedly interacts with its own genomelinked viral protein (VPg) and coat protein (CP) (Hong et al., 1995;Li et al., 1997). An interaction between the CP and the helper-component protease (HC-Pro) of Zucchini yellow mosaic virus has also been reported (Peng et al., 1998). The nuclear inclusion protein a (NIa) and cylindrical inclusion proteins have also been reported to interact with each other (Guo et al., 2001). Such interactions may be important for virus replication, virus movement and insect transmission (Blanc et al., 1997;Rojas et al., 1997;Carrington et al., 1998). Recently, we applied the yeast two-hybrid system (YTHS) to identify protein interactions among ten viral proteins generated from the SMV polyprotein (Kang et al., 2004). Here, we used the YTHS to identify key amino acid sequences or domains responsible for the self-interaction of SMV CP.Four deletion mutations were introduced into SMV-G7H CP (Fig. 1a) to define regions required for CP self-interaction. Truncated clones of the CP were fused downstream of both GAL4-DBD (pAS2-1) and GAL4-AD (pACT2; Clontech). The yeast two-hybrid plasmids pAS2-1 and pACT2, host strain AH109 (MATa, gal4D, gal80D), all media, buffers and methods for the yeast two-hybrid assay were adapted from the Matchmaker System 2 (Clontech). Proteins expressed by yeast cells were extracted (Yeast Protocol Handbook, Clontech) and verified by using SDS-PAGE followed by immunoblot with GAL4 antibodies (1 : 250 dilution; Santa Cruz Biotechnology) using an ECL kit (Amersham Biosciences). All truncated CP mutants were detecte...

Structure of Flexible Filamentous Plant Viruses

“…Most experiments were carried out using the G7 strain, but fiber diffraction experiments used both strains, and because of the better orientation and higher quality of the diffraction data, data from the G6 strain were used in the analysis. The coat proteins of the two strains differ at only 3 amino acid residues (33), and the substitutions are all conservative and within 20 residues of the N terminus, known to be located at the outer surface of the virion and probably disordered (5,56). PVX was purified and stored as previously described (52).…”

Soybean mosaic virus: a successful potyvirus with a wide distribution but restricted natural host range

“…The KLSC and PTK motifs in HC‐Pro, and the DAG motif in CP, have been confirmed to be involved in SMV aphid transmission (Jossey et al ., ; Seo et al ., ). Additional amino acid sequences near the carboxyl terminus of HC‐Pro and the putatively exposed carboxyl terminus of CP have been implicated in aphid transmissibility of SMV (Jossey et al ., ; Kang et al ., ; Seo et al ., ).…”

Architecture and Assembly of Virus Particles

“…For instance, using expression in E. coli, Jagadish et al (1993) showed that Arg 194 and Asp 238 of JGMV are required for the assembly process but were not necessarily involved in forming the predicted salt bridge. The C-terminal domain of SMV CP is required for CP-CP self-interaction (Kang et al, 2006). The C-terminal domain of SMV CP is required for CP-CP self-interaction (Kang et al, 2006).…”