Abstract
Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.
Highlights
Replication of RNA plant viruses is performed by membranebound complexes consisting of virus- and host-encoded proteins [1] where viral RNA genome multiplication requires specific interaction between the viral RNA-dependent RNA polymerase (RdRP) and its cognate RNA
By analogy with the closely related betanecrovirus, Beet black scorch virus (BBSV) [7], and TNVDH [6], three internal open reading frames (ORFs) encode small proteins that are required for viral cell-to-cell movement and the 3'-proximal ORF encodes the virus capsid protein (CP) which appears to be located in the cell nucleus [8]
The RdRP products were analysed by poly acrylamide gel electrophoresis (PAGE), which resulted in the production of two major bands for each template
Summary
Replication of RNA plant viruses is performed by membranebound complexes consisting of virus- and host-encoded proteins [1] where viral RNA genome multiplication requires specific interaction between the viral RNA-dependent RNA polymerase (RdRP) and its cognate RNA. As occurs in most members of the family Tombusviridae, TNV-D ORF 2 must be translated from the genomic RNA by readthrough of the leaky stop codon of ORF 1 and, p82 contains the sequence of p22 at the N-terminus while its C-terminal portion contains the signature motifs of RdRPs [9,10]. These motif sequences are present in many polymerases in the ‘palm subdomain’ which is composed of a four-stranded antiparallel beta-sheet with two alpha-helices and includes the motifs A, B and C.
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