The RNA genome of tobacco etch virus (TEV) encodes a large polyprotein precursor that is processed to mature proteins by virus‐specific proteinases. Cleavage sites located within the carboxyl‐terminal two‐thirds of the polyprotein are processed by a TEV‐encoded 49 kd proteinase, while the enzyme(s) responsible for cleaving the remaining sites has not been found. In this study, a second TEV‐encoded proteinase has been identified based on cell‐free expression of defined RNA transcripts. The boundaries of this proteinase have been delineated by deletion analysis and site‐directed mutagenesis. The proteolytically active domain has been localized to the carboxyl‐terminal half of the 56 kd aphid‐transmission helper component. A cleavage site that is recognized by this proteinase has been identified in the polyprotein adjacent to the carboxyl‐terminus of the enzyme, and the proteinase appears to cleave by an autocatalytic mechanism. Proteolysis in vitro occurs between a Gly‐Gly dipeptide as determined by radiochemical sequencing at the amino‐terminus of the proteolytic product.

No abstract

No abstract

No abstract

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus‐encoded proteinase at five different sites between the dipeptides Gln‐Ser or Gln‐Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence…Glu‐Xaa‐Xaa‐Tyr‐Xaa‐Gln‐Ser or Gly…. We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site‐directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell‐free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.

The tobacco etch virus 27-kDa nuclear inclusion a (NIa) proteinase was expressed in Escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. Catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. The active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. This conversion was dilution independent and thought to be intramolecular. Isolation of the approximately 2-kDa peptide cleavage product and determination of its N-terminal amino acid sequence positioned the cleavage site 24 amino acids from the carboxy-terminus of the proteinase. A recombinant NIa proteinase lacking the C-terminal 24 amino acids was shown to possess limited activity. Kinetic analyses of cleavage of a synthetic peptide by the full-length or truncated proteinase were conducted and indicated that the Km of the truncated proteinase was approximately fourfold higher than that of the full-length form. The truncated proteinase was approximately one-twentieth as efficient in proteolysis of the test peptide substrate as the full-length form.

No abstract

Haploid leaf tissue of tobacco cultivars K326 and K149 was transformed with severa1 transgenes containing cDNA of the potato virus Y (PVY) coat protein (CP) open reading frame (ORF). The various transgenes containing the PVY CP ORF sequence produced (1) the expected mRNA and CP product, (2) an mRNA rendered untranslatable by introduction of a stop codon immediately after the initiation codon, or (3) an antisense RNA that was untranslatable as a result of the incorrect orientation of the PVY CP ORF behind the transcriptional promoter. Homozygous doubled haploid (DH) (diploid) plants were generated, and selfed progeny from these plants were examined. Reslstance was virus specific, functioning only against PVY. An inverse correlation between transgene-derived PVY transcript steady state levels and resistance was generally noted with lines expressing the untranslatable sense version of the PVY CP ORF. A collection of DH lines, derived from a single transformation event of a common haploid plant and isogenic for the PVY transgenes expressing untranslatable sense RNA, displayed different levels of PVY resistance. Lines with actively transcribed, methylated transgene sequences had low steady state levels of transgene transcript and a virus-resistant phenotype. These results are discussed within the context of sense suppression in plants.