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 subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained 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 displayed wild-type viral protein synthesis and host shut-off kinetics.
Piccone
M E
ME
United States Department of Agriculture, Agricultural Research Service, Greenport, New York 11944, USA.
Zellner
M
M
Kumosinski
T F
TF
Mason
P W
PW
Grubman
M J
MJ
D000818:Animals; D005537:Aphthovirus / Q000201:enzymology* / Q000502:physiology; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D011499:Protein Processing, Post-Translational; D014779:Virus Replication / Q000235:genetics
D010450:Endopeptidases; C088747:leader proteinase, foot-and-mouth disease virus