Cleavage site mutations in the encephalomyocarditis virus P3 region lethally abrogate the normal processing cascade.

Site-specific mutations within the proteinase 3C-dependent P3 region cleavage sequences of encephalomyocarditis virus have been constructed. The mutations altered the normal QG cleavage site dipeptide pairs of the 2C/3A, 3A/3B, 3B/3C, and 3C/3D junctions into QV, QC, QF, QY, and RG sequences. When translated in vitro in the context of full-length viral polyproteins, all mutations blocked endogenous 3C-mediated processing at their engineered sites and produced stable forms of the expected viral P3 precursors that were also resistant to cleavage by exogenously added recombinant 3C. Relative to wild-type viral sequences, each mutant form of P3 had a somewhat different ability to mediate overall polyprotein processing. Mutations at the 2C/3A, 3A/3B, and 3B/3C sites, for example, were generally less impaired than 3C/3D mutations, when the cleavage reactions were quantitated with cotranslated L-P1-2A precursors. A notable exception was mutant 3B3C(QG3RG), which provedfarlessactivethansiblingmutants3B3C(QG3QF)and3B3C(QG3QV),afindingthatpossiblyimplicates thissegmentintheproperfoldingofanactive3C.WhentransfectedintoHeLacells,allmutantsequenceswere lethal, presumably because of the reduced L-P1-2A processing levels or reduced RNA synthesis capacity. However, when specifically tested for the latter activity, all mutations except those at the 3C/3D cleavage site were indeed able to initiate and perpetuate viral RNA replication in transfected cells, albeit to RNA accumulationlevelslowerthanthoseproducedbywild-typesequences.Thetransfectioneffectscouldbemimickedwith cell-free synthesized proteins, in that translation samples containing locked 3CD polymerase precursors were catalyticallyinactiveinpoly(A)-oligo(U)-dependentassays,whileallothermutantprocessingsamplesinitiated detectable RNA synthesis. Surprisingly, not only did the 3B/3C mutant sequences prove capable of directing RNA synthesis, but the viral RNA thus synthesized could be immunolabeled and precipitated with 3C-specific monoclonal antibody reagents, indicating an unexpected covalent attachment of the proteinase to the RNA product whenever this cleavage site was blocked. Picornavirus RNA genomes encode a large polyprotein which is proteolytically processed in cotranslational and posttranslationalreactionsbyseveralself-encodedreactivecenters. The majority of cleavages are catalyzed by one specific proteinase, 3C, an unusual cysteine-reactive, chymotrypsin-like sequence located in the carboxyl third of the polyprotein. During translation, 3C is synthesized in an enzymatically active form, and even while still part of the nascent polyprotein, it rapidly and efficiently catalyzes an ordered series of monomolecular and bimolecular scissions within its parental molecule to release mature viral proteins and precursor combinations. The consensus 3C cleavage sites in most picornavirus polyproteins are at Gln-Gly dipeptide pairs, but not all conforming viral sequences are always cleaved, and in many virus strains, alternative combinations like Gln-Ser, Gln-Ala, GluGly,orGlu-Seroccuratthenaturalsites(6).Experimentswith