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.

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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 genome of tobacco etch virus contains a single open reading frame with the potential to encode a 346-kilodalton (kDa) polyprotein. The large polyprotein is cleaved at several positions by a tobacco etch virus genome-encoded, 49-kDa proteinase. The locations of the 49-kDa' proteinase-mediated cleavage sites flanking the 71-kDa cytoplasmic pinwheel inclusion protein, 6-kDa protein, 49-kDa proteinase, and 58-kDa putative polymerase have been determined by using cell-free expression, proteolytic processing, and site-directed mutagenesis systems. Each of these sites is characterized by the conserved sequence motif Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly (in which cleavage occurs after the Gln residue). The amino acid residue (Gln) predicted to occupy the-1 position relative to the scissile bond has been substituted, by mutagenesis of cloned cDNA, at each of four cleavage sites. The altered sites were not cleaved by the 49-kDa proteinase. A series of synthetic polyproteins that contained the 49-kDa proteinase linked to adjoining proteins via defective cleavage sites were expressed, and their proteolytic activities were analyzed. As part of a polyprotein, the proteinase was found to exhibit cis (intramolecular) and trans (intermolecular) activity.

We have developed a vector (pTL-8) to facilitate cell-free expression of cloned protein-coding sequences (1). The 5' non-coding and initial coding sequences of the tobacco etch virus (TEV) RNA genome were inserted downstream from an SP6 promoter, allowing foreign coding sequences to utilize the translational initiation properties of the TEV leader for cell-free translation from synthetic transcripts. Efficient and accurate initiation of translation occurs without the need for 5 capping procedures (1). A family of vectors has been developed which increases the applicability of pTL-8 and which allows single-stranded DNA production for oligonucleotide-directed mutagenesis.