Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

Abstract

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the mature proteins. Cell-free translation of foot-and-mouth disease virus (FMDV) RNA in a rabbit reticulocyte system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.

Distinctive features of foot-and-mouth disease virus, a member of the picornavirus family; aspects of virus protein synthesis, protein processing and structure

“…In contrast the inhibition of host translation is observed with FMDV infection and this is accompanied by cleavage of p220 (Devaney et al, 1988). It has been shown that this effect is induced by the L protein as indicated above (Vakharia et al, 1987;Belsham and Brangwyn, 1990;Medina et al, 1993). It has been reported that the p220 cleavage products induced by FMDV infection differ in mobility from those induced by PV infection Kleina and Grubman, 1992).…”

“…In PV it is clear that at least some of the cleavages (those within the capsid precursor) require the precursor 3CD (Jore et al, 1988;Ypma-Wong et al, 1988), the 3C protein alone is insufficient. However in FMDV all the capsid protein processing events to produce lAB, 1C and 1D can be mediated by 3C alone (Vakharia et al, 1987;Ryan et al, 1989;. The details of P2 and P3 processing have not yet been fully elucidated in FMDV or other picornaviruses.…”

Foot-and-Mouth Disease Virus 3C Protease Cleaves NEMO To Impair Innate Immune Signaling

“…Protease activity governs the ability of FMDV 3C pro to suppress dsRNA-induced IFN response. As with the 3C pro of other picornaviruses, FMDV 3C pro is a cysteine proteinase responsible for most cleavages within the viral polyprotein (31,48). For FMDV 3C pro , residues His-46 and Cys-163 are part of the catalytic triad and His-181 is part of the binding pocket (6,24).…”

Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus

“…Plasmid pLb-VP4'-3D' was derived from pTLP123 (Vakharia et al, 1987) by digestion with NcoI and religation (Fig. 1).…”

“…Transcripts from Ncol or BamHI linearized pLb-VP4'-3D' or BamHI linearized pLb-T7 were synthesized with T7 RNA polymerase and the mMESSAGE, mMACHINE capping kit (Ambion). Approximately 0.5-1 /xg of RNA from transcription reactions or 1 /zg FMD A12 virion RNA was used in a rabbit reticulocyte lysate in vitro translation system as previously described (Vakharia et al, 1987;Bablanian and Grubman, 1993). E-64 was purchased from Boehringer-Mannheim.…”