Biochemical and mutational analysis of a plant virus polyprotein cleavage site

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.