IDENTIFICATION OF THE ACTIVE-SITE RESIDUES OF THE L-PROTEINASE OF FOOT-AND-MOUTH-DISEASE VIRUS

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a submit of the cap-binding prote in complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this informa tion, we have investigated the active-site residues by introducing spe cific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cia activity at the L/VP4 clea vage site, trans activity on a truncated L-P1 substrate, and p220 clea vage activity. Mutations of Cys-23 to Ser or His-110 to Leu resulted i n enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-lengt h FMDV cDNA, and RNA transcripts derived from these cDNAs were transla ted in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within p1, and virus ob tained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displ ayed wild-type viral protein synthesis and host shut-off kinetics.