Tobacco etch virus (TEV) encodes three proteinases that catalyze processing of the genome-encoded polyprotein.
The P1 proteinase originates from the N terminus of the polyprotein and catalyzes proteolysis between
itself and the helper component proteinase (HC-Pro). Mutations resulting in substitution of a single amino
acid, small insertions, or deletions were introduced into the P1 coding sequence of the TEV genome. Deletion
of the N-terminal, nonproteolytic domain of P1 had only minor effects on virus infection in protoplasts and
whole plants. Insertion mutations that did not impair proteolytic activity had no measurable effects regardless
of whether the modification affected the N-terminal nonproteolytic or C-terminal proteolytic domain. In
contrast, three mutations (termed S256A, F, and Δ304) that debilitated P1 proteolytic activity rendered the
virus nonviable, whereas a fourth proteinase-debilitating mutation (termed C) resulted in a slow-infection
phenotype. A strategy was devised to determine whether the defect in the P1 mutants was due to an inactive
proteinase domain or due simply to a lack of proteolytic maturation between P1 and HC-Pro. Sequences coding
for a surrogate cleavage site recognized by the TEV NIa proteinase were inserted into the genome of each
processing-debilitated mutant at positions that resulted in NIa-mediated proteolysis between P1 and HC-Pro.
The infectivity of each mutant was restored by these second-site modifications. These data indicate that P1
proteinase activity is not essential for viral infectivity but that separation of P1 and HC-Pro is required. The
data also provide evidence that the proteinase domain is involved in additional, nonproteolytic functions
