Proteolytic cleavage of encephalomyocarditis virus capsid region substrates by precursors to the 3C enzyme

Abstract

Picornavirus protease 3C is normally released from its P3 precursor by two successive self-cleavage reactions. The free enzyme can then catalyze most of the remaining processing events within the viral polyprotein. To investigate the role of the 3C precursors in the processing cascade, we constructed cDNA clones which expressed genetically altered forms of the encephalomyocarditis P3 region in vitro. Site-specffic substitutions were introduced into the Gln-Gly residues at the 3B-3C and 3C-3D junctions, and the resulting proteins were tested for their ability to self-process and to catalyze cleavage of viral capsid precursors in cell-free protease assays. We determined that three P3 region precursor proteins (3ABC, 3CD, and P3), harboring inactive cleavage sites, were as active as the free enzyme (3C) in processing assays with capsid substrates. Further, we found that in addition to the naturally occurring Gln-Gly and Gln-Ser amino acid pairs, the encephalomyocarditis 3C enzyme was able to process GIn-Cys but not Gln-Thr, Gln-Ile, Gin-Tyr, Arg-Gly, or Leu-Gly combinations when these residues were substituted into normal cleavage site contexts.

Identification and characterization of a 3C-like protease from rabbit hemorrhagic disease virus, a calicivirus

“…Our expression studies showed that the protease retains activity if the putative polymerase domain is removed. It should be noted, however, that we never detected processing at the protease-polymerase boundary, an event observed for picornaviruses when similar constructs were used (17,36). To clarify this issue further, functional studies and size estimation of the protease in infected cells are needed.…”

The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase

“…Two QG dipeptides proposed to be cleaved in IBV were substituted in MHV by QC in one case, and by KR dipeptide in another (Table 1). Substitution of a C (unlike several other residues) for G in a cleavage site for encephalomyocarditis virus protease did not abolish processing in an in vitro system (Parks et a/., 1989). Dibasic dipeptides are cleaved in the polyproteins of flaviviruses (Strauss and Strauss, 1988).…”

Rapamycin and Wortmannin Enhance Replication of a Defective Encephalomyocarditis Virus

“…To distinguish between these two possibilities, wild-type, ⌬2A and ⌬2A* EMCV RNAs were translated in a rabbit reticulocyte lysate for a short period (25 min) in the absence or presence of purified 3C pro from mengovirus. In the absence of 3C pro , the polypeptides L-P1-2A and 2C were synthesized, but not 3C pro or 3ABC, which are responsible for polyprotein processing (23,39,41,54), or any other P3-derived polypeptides (Fig. 7).…”