Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein

Abstract

Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.

Peptide-mediated broad-spectrum plant resistance to tospoviruses

“…Consistent with the general notion of the multifunctionality of viral coat proteins (14), the TSWV N protein was found to be involved in a number of molecular interactions, such as homopolymerization (15), packaging of the viral genomic RNA (16), and interaction with NSm, the TSWV movement protein (17). The molecular dissection of homotypic interaction of TSWV N protein revealed the existence of two interaction domains and identified amino acids conserved among the tospoviruses that are essential for the interaction (15).…”

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

“…Similar competition assays by using purified TSWV N protein have revealed a virtually irreversible RNA binding of this protein (7). The fact that NSm binds RNA much less efficiently suggests a transient NSm-RNA interaction during the transport process.…”

Expression, purification, crystallization and preliminary X-ray crystallographic study of the nucleocapsid protein ofTomato spotted wilt virus

“…Interestingly, the sequence and structure of N proteins are highly conserved within a genus but differ between genera. For a full understanding of the genome encapsidation mechanism of the genus Tospovirus, the structure of the N protein from TSWV is indispensable, although the domains or aminoacid residues contributing to RNA binding or oligomer formation have been suggested by biochemical analyses (Richmond et al, 1998;Uhrig et al, 1999). Here, we report the crystallization and preliminary X-ray crystallographic analysis of the TSWV N protein to gain further insight into its oligomeric state and the mechanism of RNA binding.…”

“…The S RNA segment encodes the nucleocapsid (N) and NSs proteins, the M RNA segment encodes glycoproteins (Gn and Gc) and NSm proteins, and the L RNA segment encodes the RNA-dependent RNA polymerase (RdRp) (Adkins, 2000). The numerous N proteins encapsidate viral genome RNAs and, in association with a few RdRps, constitute vRNP (viral ribonucleoprotein) complexes (Uhrig et al, 1999;Richmond et al, 1998). These vRNP complexes perform genome RNA replication and viral mRNA transcription.…”