Investigations on the Tobacco Necrosis Virus D p60 replicase protein

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.

Figure 1. Schematic representation of TNV-D genomic and sub-genomic RNAs and expression and purification of recombinant MBP-TNV-D p60.

In panel (a) open boxes correspond to predicted ORFs including ORFs 1 and 2 which encode proteins p22 and p82 respectively. Protein p60 is predicted from the sequence downstream of the leaky UAG amber stop codon [3]. The genomic and sub-genomic TNV-D RNAs encoding the virus proteins and their sizes are shown ABOVE the gene map. Panel B shows expression and purification of recombinant MBP-TNV-D p60 from E. coli. The MBP-p60 fusion protein was expressed at 25°C for 4 h following induction with 0.5 mM IPTG. Proteins were analysed by 10% SDS-PAGE and subjected to Coomassie blue staining. Lane 1 contains protein markers with their molecular masses (in kDa) indicated on the left. Lane 2 contains highly purified MBP-p60 following Superose 6 10/300 column purification.

Figure 2. Kinetics of RdRP activity of MBP-TNV-D p60 and effects of S1 nuclease treatment on radiolabelled RNA products.

Panel (a) shows representative denaturing gel analysis of radiolabelled RNA products synthesised at 25°C (RT) by in vitro transcription using satC- RNA as the template over a 16 h time course. The data is also shown in graphical form quantified by AIDA following phosphorimager analysis. Panel (b) shows the effects of S1 nuclease treatment on radiolabelled RNA products synthesised at 25°C. Products synthesised following 1, 2 or 3 h incubation were untreated (lanes 3, 5 and 7 respectively) or treated with S1 nuclease (lanes 4, 6 and 8 respectively). Radiolabelled products were stained with ethidium bromide in the left hand panel which also shows the template satC- RNA used in these reactions, untreated (lane 1) or treated (lane 2) with S1 nuclease, which is arrowed. Phosphorimaging of the gel is shown in the right hand panel.

Figure 3. Analysis of RNA-binding properties of MBP-TNV-D p60 in vitro.

Representative electrophoretic mobility shift assay (EMSA) showing interactions between recombinant p60 and ss RNA. The [32P]-labelled satC- RNA probe (28.4 pmol) was incubated with no protein (lane 1) or with MBP-p60 fusion protein at 1, 3, 5, 7, 9, 11 or 13 μg concentration (lanes 2-8, respectively). The unbound (free) RNA probe and the shifted (bound) RNA complexes are marked on the right. The data is expressed in graphical form following phosphorimager analysis and quantification by AIDA.

Figure 4. Effect of mutations to MBP-p60 on the synthesis of STNV-C RNA.

The upper tier in panel (a) shows 10% SDS-PAGE analysis of the expressed proteins, the lower tier shows 5% denaturing polyacrylamide-8 M urea gel analysis of radio-labelled STNV-C dsRNA. Individual mutations are shown above the respective lanes. Radioactivity was quantified by AIDA and expressed as a percentage of the radio-labelled dsRNA produced by wt MBP-p60 in four parallel assays and the data presented as histograms in panel (b).

Figure 5. Effect of mutations in the TNV-D p60 domain on RNA replication in Nicotiana benthamiana.

Total RNA extracted from inoculated leaves in panel (a) and systemically infected leaves in panel (b) following inoculation with wt- and mutant RNA transcripts was fractionated by agarose gel electrophoresis and analysed by northern blot hybridisation. TNV-D genomic RNA and two sub-genomic RNAs are indicated by arrows. The tobacco 25S and 18S rRNAs were stained with ethidium bromide for loading purposes. Radioactivity was quantified by AIDA and the results with inoculated and systemically infected leaves are presented as histograms in panels (c) and (d) respectively.