Homotypic interaction and multimerization of nucleocapsid protein of tomato spotted wilt tospovirus: identification and characterization of two interacting domains

Abstract

The nucleocapsid protein (N) of tomato spotted wilt tospovirus (TSWV) plays a central role in the viral life cycle. With the aid of the yeast two-hybrid system and surface plasmon resonance analysis, homotypic interaction and multimerization of the N protein was detected. Analysis of deletion mutants identified two binding regions in the protein, located at the N terminus (amino acids 1-39) and the C terminus (amino acids 233-248), respectively, implying a "head-to-tail" interaction of the N terminus with the C terminus to form a multimeric chain. Further characterization of the binding domains was performed by site-directed mutagenesis. Two phenylalanines (F242 and F246) highly conserved in the N proteins within the Tospovirus genus were shown to play a crucial role in the interaction.

Figure 1

Cells were plated onto selective medium lacking histidine and supplemented with 25 mM 3-aminotriazole (a) and tested for β-galactosidase activity by using X-Gal on a filter lift assay (b). Cells were transformed with the following: 1, pBD-N only; 2, pAD-N only; 3, pBD-SNF1/pAD-SNF4; 4, pAS2/pAD-N; 5, pBD-N/pACT2; 6, pBD-SNF1/pAD-N; 7, pBD-N/pAD-SNF4; or 8, pBD-N/pAD-N.

Figure 2

SPR of the interaction of TSWV N protein subjected to a SA5 sensor chip that had been preloaded with N protein. The sensor chip was alternately rinsed with a 30 nM protein solution (at 190, 550, and 860 sec) and the respective buffer (at 0, 370, and 730 sec).

Figure 3

Deletional and mutational constructs generated for the yeast two-hybrid system to investigate homotypical interaction and interaction with wild-type N protein. Numbers of amino acid residues are indicated. A red A indicates schematically where alanine was substituted for phenylalanine.

Figure 4

Homotypic interaction of wild-type N protein (B) and interaction of C- and N-terminally truncated proteins (C). The strength of the interaction was determined by assaying the β-galactosidase activity with ONPG. Aliquots containing 10⁷ cells of an overnight culture were spotted onto a membrane and stained with X-Gal. Empty vector constructs and SNF1/SNF4 were used as controls (A).

Figure 5

C-terminal part of tospoviral N proteins. Multiple sequence alignment using pileup [Wisconsin Package Version 9.1, Genetics Computer Group (GCG), Madison, WI]. Dots indicate amino acid residues identical to those in TSWV-L3; asterisks indicate residues F242 and F246 that have been mutated to alanine; and hyphens denote gaps. GenBank, EMBL, PIR, Swiss-Prot accession numbers: TSWV-L3 (tomato spotted wilt virus), P26001; TSWV-BR-01 (tomato spotted wilt virus), 420617; GRSV-SA-05 (groundnut ring spot virus), P36294; TCSV-12851 (tomato chlorotic spot virus), P36293; INSV-NL-07 (impatiens necrotic spot virus), 60490; INSV-Beg (impatiens necrotic spot virus), 310991; WSWV-W (watermelon silver mottle virus), 1019831; PBNV-40687 (peanut bud necrosis virus), 881480; and IYSV-60456 (iris yellow spot virus), 2228768.

Figure 6

Interaction of full-length and N-terminally truncated proteins in which F242 and/or F246 has been mutated to alanine. Constructs were tested for homotypic interaction and interaction with wild-type N protein (wt-N) (A). The strength of the interaction was assayed by a β-galactosidase activity test using ONPG. Additionally, interaction between a set of deletional or mutational constructs was determined (B).