The S RNA Segment of Tomato Spotted Wilt Virus has an Ambisense Character

Abstract

The complete nucleotide sequence of the S RNA of tomato spotted wilt virus (TSWV) was determined. The RNA is 2916 nucleotides long and has an ambisense coding strategy. The sequence contains two open reading frames (ORFs), one in the viral sense which encodes a protein with a predicted Mr of 52"4K and one in the viral complementary sense which encodes the viral nucleocapsid protein of Mr 28"8K. Both proteins are expressed by translation of two subgenomic RNA species that possibly terminate at a long stable hairpin structure, located at the intergenic region. The structure of this RNA segment resembles that of the arthropod-borne phleboviruses (family Bunyaviridae). The absence of significant sequence homology between TSWV and bunyaviruses infecting animals suggests that TSWV should be considered as a representative of a new genus within the Bunyaviridae.

The movement protein NSm of tomato spotted wilt tospovirus (TSWV): RNA binding, interaction with the TSWV N protein, and identification of interacting plant proteins

“…Apparent homogeneity of the eluted protein was evaluated by SDS͞PAGE, and these samples were dialyzed against 300 mM NaCl͞50 mM Tris⅐HCl, pH 8.0. (His) 6 -tagged TSWV N protein was expressed and purified as described previously (8).…”

“…The NSm gene [TSWV isolate L3 (24)] was cloned into the vector pET-28a (Novagen) and expressed with an N-terminal (His) 6 -fusion in E. coli HMS174(DE3). To enhance the NSm translation, the argU gene encoding a rare tRNA arg4 [(25); S. Leclair, personal communication] was coexpressed in Escherichia coli.…”

A protoplast system for studying tomato spotted wilt virus infection.

“…The percentage of protoplasts infected was estimated by immunofluorescence, using an antiserum against NSs, a nonstructural protein only produced after replication of the genome (de Haan et al, 1990), and a reliable indicator of infection. The proportion of infected protoplasts was calculated as numbers of NSs-positive cells relative to total numbers of living cells.…”

“…other two (S and M) RNAs to be ambisense [de Haan et al (1990) and , respectively]. In total six viral proteins appear to be specified by the genome of TSWV.…”

“…The M RNA encodes, in its viral (v) strand, a common precursor to both envelope glycoproteins and, in its viral complementary (vc) strand, a nonstructural protein, NSm, implicated in cell-to-cell movement of the virus (Kormelink et al, a, 1994Storms et al, 1995). The (smallest) S RNA encodes the N protein in the vc strand and an additional nonstructural protein, NSs, whose function remains unknown, in the v strand (de Haan et al, 1990 ;Kormelink et al, 1991).…”