Identification of the active-site residues of the L proteinase of foot-and-mouth disease virus.

摘要: The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and of translation initiation factor p220, a subunit cap-binding complex. It has been suggested that this proteinase homology to papain-like family cysteine proteinases, from information, we have investigated active-site residues by introducing specific mutations into L gene. Mutations Cys-23 Ala or His-120 Leu resulted enzymes lackedcis activity L/VP4 site, trans on truncated L-P1 substrate, p220 activity. Ser His-110 retained some allcisactivity had reduced cleavage. These were introduced separately full-length FMDV cDNA, RNA transcriptsderivedfromthesecDNAsweretranslatedinacell-freesystemandtransfectedintocells.TheC23S mutant inefficiently cleaved within P1, obtained transfected cells reverted wild type. H110L almost as well wild-type enzyme, recovered mutation displayed viral synthesis host shut-off kinetics. Foot-and-mouthdiseasevirus(FMDV)virionRNAistranslatedintoapolyproteinwhichisprocessed,duringitssynthesis, four primary products, L, P2, P3. Initiation begins either two in-frame AUG codons, approximately 1,100 bases 59 end RNA, resulting proteins, Lab Lb, differing their amino termini 28 acids (24). smaller major species synthesized infected cell-free system. Both proteins autocatalytically cleave themselves polyprotein (26) complex eIF-4, 59end capped mRNAs (3, 14, 20). Cleavage component results most cell (5), but translation, which occurs cap-independent mechanism, not impaired. Analysis acid sequence data thiol protease related papain proteases (7). We shown E-64, inhibitor proteases, including (10), blocks system an uncharged analog, E-64d, (15). Furthermore, demonstrated E-64d assembly and, consequence, reduces yield, suggesting compound might be effective agent against footand-mouth disease. inserted Lb gene plasmid under control T7 promoter expressed hascisandtranscleavage