The JV 3C protease mutant was created by point mutation of the critical TGT encoded cysteine residue, within the GDCG active site motif, to a GGT encoded glycine residue by mutagenic overlap PCR using Bio-X-Act DNA polymerase. Three rounds of amplification using the JV full length cDNA clone as template were used to generate the final mutant protease cassette. Round 1 used the primers JV F1 (59-CGTCTCAGGGTTGATACT-39) and JV Mut 1 (59-GCAACCACCGTCACCAG-39), yielding a 222 bp amplicon (point mutation nucleotide shown in bold). Round 2 used the primers JV Mut 2 (59-CTGGTGACGGT-GGTTGC-39) and JV R2 (59-TTCCTGGGAGGAACAAGTT-39), yielding a 651 bp amplicon. Amplicons generated in rounds 1 and 2 were pooled to serve as template for round 3 using the primers JV NF (59-ATGTCAACCACCACCAGC -39) and JV NR (59-AAGGGCTCCGGTGAAGG-39). This cassette contained two BclI restriction sites flanking the 3Cprotease active site, as also found in the wild-type full length clone. Restriction digest using BclI was used to remove the appropriate wild-type cassette from the JV full length clone. The mutant cassette was also digested with BclI prior to ligation to the BclI-digested JV full length clone. The ligated DNA was used to transform E.coli Top10, and was designated JV 3C mut .