Functional analysis of the interaction between VPg-proteinase (NIa) and RNA polymerase (NIb) of tobacco etch potyvirus, using conditional and suppressor mutants

Abstract

The tobacco etch potyvirus (TEV) RNA-dependent RNA polymerase (NIb) has been shown to interact with the proteinase domain of the VPg-proteinase (NIa). To investigate the significance of this interaction, a Saccharomyces cerevisiae two-hybrid assay was used to isolate conditional NIa mutant proteins with temperature-sensitive (ts) defects in interacting with NIb. Thirty-six unique tsNIa mutants with substitutions affecting the proteinase domain were recovered. Most of the mutants coded for proteins with little or no proteolytic activity at permissive and nonpermissive temperatures. However, three mutant proteins retained proteolytic activity at both temperatures and, in two cases (tsNIa-Q384P and tsNIa-N393D), the mutations responsible for the ts interaction phenotype could be mapped to single positions. One of the mutations (N393D) conferred a ts-genome-amplification phenotype when it was placed in a recombinant TEV strain. Suppressor NIb mutants that restored interaction with the tsNIa-N393D protein at the restrictive temperature were recovered by a two-hybrid selection system. Although most of the suppressor mutants failed to stimulate amplification of genomes encoding the tsNIa-N393D protein, two suppressors (NIb-I94T and NIb-C380R) stimulated amplification of virus containing the N393D substitution by approximately sevenfold. These results support the hypothesis that interaction between NIa and NIb is important during TEV genome replication.

FIG. 1

Maps of the TEV genome, NIa, and sequences subjected to random mutagenesis. The TEV genome is represented at the top, with the polyprotein coding region indicated in grey. The names of several individual TEV proteins are given above the map. The sequences coding for cleavage sites are shown as short vertical lines within the coding region. The NIa sequence is represented in the expanded diagram, with the N-terminal VPg and C-terminal proteinase (thick line) domains indicated. Arrows indicate positions of suboptimal internal cleavage sites within NIa. The VPg attachment site (Tyr62) is indicated. The sequences independently mutagenized in the two libraries are shown at the bottom. Library 1 was composed of two sublibraries as represented by the two offset horizontal lines.

FIG. 2

Examples of β-galactosidase screens for temperature sensitivity of the NIa-NIb interaction in yeast. Colonies were plated in triplicate on each of two nitrocellulose filters incubated on interaction-nonselective medium, grown at 20 or 30°C, and processed for the β-galactosidase colorimetric assay. Results with two tsNIa mutant proteins (tsNIa-5b and tsNIa-26a) are shown along with those of positive-interaction and negative controls. wt, wild type.

FIG. 3

Immunoblot assay for self-processing of selected tsNIa mutants in E. coli. (A) Cells were grown at 20°C, induced by addition of IPTG, cultured for an additional 4 h, and harvested. Total SDS-soluble protein extracts were subjected to immunoblot analysis with a monoclonal antibody cocktail specific for the proteinase domain of NIa. The cells contained an empty expression vector, pET23d(+), or a vector expressing the NIa-related protein indicated. (B to D) Time course analysis of self-processing at 20 and 30°C in E. coli with wild-type NIa (wtNIa) (B), tsNIa-Q384P (C), or tsNIa-N393D. Samples were withdrawn at the times postinduction (in hours) indicated below the gels and subjected to immunoblot assay with the antibody cocktail used in panel A. The positions of full-length NIa, the proteinase domain of NIa (NIaPro), and the proteinase domain lacking the C-terminal 24 residues (NIaPro-24) are shown next to each panel.

FIG. 4

Quantitative β-galactosidase assay for interaction in the yeast two-hybrid assay. Liquid cultures were grown in duplicate sets under interaction-nonselective conditions at 20 or 30°C. The yeast strains expressed the NIb fusion protein and empty cloning vector (pACT-2), wild-type NIa (wtNIa), or the mutant NIa’s indicated. β-Galactosidase assays were done with three independent cultures for each strain at both temperatures, and the means ± standard deviations are plotted.

FIG. 5

Amplification of TEV-GUS genomes containing tsNIa mutant alleles in protoplasts. (A) Time course analysis of parental TEV-GUS, replication-defective TEV-GUS/VNN, and TEV-GUS/tsNIa-N393D at 20 and 30°C. Each data point represents the mean of results from three independent inoculations done simultaneously with the same batch of protoplasts. (B) Relative levels of amplification of control and tsNIa mutant TEV-GUS genomes in protoplasts at 20 and 30°C. Parental TEV-GUS (wild type [wt]) was used as the 100% standard at both temperatures. Each bar represents the mean relative amplification level (± standard deviation) at 48 h p.i. from six independent infections.

FIG. 6

Isolation and analysis of NIb suppressor mutant proteins that restore interaction with tsNIa-N393D at 30°C in the yeast two-hybrid system. (A) Quantitative β-galactosidase assays of yeast cultures containing pACT-tsNIa-N393D and pAS-NIb alleles with the indicated mutations. Each bar represents the mean activity (± standard deviation) (in Miller units) from three independent cultures. (B) Relative levels of stimulation of amplification of TEV-GUS/tsNIa-N393D genomes containing the NIb suppressor alleles indicated in protoplasts at 48 h p.i. Amplification was calculated by using TEV-GUS/tsNIa-N393D, with results for wild-type NIb (wtNIb) as the relative standard being equal to 1. Each bar represents the mean (± standard deviation) of results from three independent infections.

FIG. 7

Amplification of TEV-GUS/tsNIa-N393D mutant genomes containing NIb suppressor alleles in protoplasts. (A) Time course analysis of parental TEV-GUS (wild type [wt]), replication-defective TEV-GUS/VNN, and TEV-GUS/tsNIa-N393D with the wild-type NIb, NIb-I94T, or NIb-C380R allele at 20 and 30°C. Each data point represents the mean of results from three independent inoculations done simultaneously with the same batch of protoplasts. (B) Relative levels of amplification of TEV-GUS mutant genomes containing the NIb suppresor alleles with wild-type NIa or the tsNIa-N393D allele in protoplasts at 30°C. Parental TEV-GUS was used as the 100% standard. Each bar represents the mean relative amplification level (± standard deviation) at 48 h p.i. from three independent infections.