49). The viral polyprotein is processed into the individual viral gene products by the viral 3C protease, itself a part of the polyprotein (50). The picornaviral 3C protease is the prototype of the new class of chymotrypsin-like cysteine proteases (49, 51, 52). It can cleave itself out of the viral polyprotein in cis and in trans, when it is expressed as part of the polyprotein or separately (53, 54).
The autocatalytic excision is not correlated with concomitant development of the proteolytic activity. The precursors of the 3C gene product already have proteolytic activity (55–58). In the viruses of the genus enterovirus the precursor 3CD (the 3D gene product constitutes the RNA-dependent RNA polymerase) shows a proteolytic activity that is distinct from that of 3C. In poliovirus, and presumably in most other enteroviruses, the proteolytic activity of the precursor 3CD is required for the efficient processing of the capsid precursor proteins (58). In hepatitis A virus the 3ABC gene product appears to be an important, proteolytically active intermediate of the polyprotein processing (57).
Palmenberg and Rueckert (59) examined the kinetics of the polyprotein processing in the picornavirus, encephalomyocarditis virus. Their data suggest that the autocatalytic excision of the 3C gene product can be a truly intramolecular event. Further evidence for an intramolecular excision of the 3C protease from poliovirus was provided by Hanecak et al. (60). Taken together, these data suggest that the autocatalytic excision of 3C from the polyprotein at both the N and C termini can be either intramolecular or intermolecular.
Once atomic resolution structures of 3C proteases became available (61–63), it was possible to develop structural models for the autocatalytic excision of the picornaviral 3C proteases. The crystal structures confirmed that the picornaviral 3C proteases are structurally related to the chymotrypsin family of serine proteases. Based on some of the unique structural details, the authors of the crystal structure papers (61–63) proposed similar models for an autocatalytic, intramolecular cleavage at the N terminus of 3C. They also agreed that it is much less obvious how an intramolecular cleavage could occur at the C terminus of 3C.
The N-terminal residues of the picornaviral 3C proteases form a stable a-helix that precedes the first strand of the N-terminal ß-barrel domain. This helix packs against the surface of the C-terminal domain of 3C. The last turn of this a-helix is formed by the residues of a highly conserved sequence motif K/RR/KNI/L (48).
Another unusual feature of the picornaviral 3C proteases is an antiparallel ß-ribbon that extends from the C-terminal ß-barrel (49). It forms an extension of the second and third ß-strands of the C-terminal domain and corresponds topologically to the methionine loop of the chymotrypsin-like serine proteases. This feature is also present in the bacterial serine proteases such as a-lytic protease and Streptomyces griseus protease B (64, 65). The recent crystal structure of a-lytic protease complexed with its prosegment (2) revealed that this feature plays an important role in the folding of the protease and in the autocatalytic, intramolecular processing of the precursor of a-lytic protease.
The structural model of an intramolecular cleavage at the N terminus of the picornaviral 3C proteases (Fig. 5) predicts that the N-terminal a-helix folds to its final conformation only after the 3C protease has cleaved its own N terminus (61–63). Before the intramolecular cleavage at the 3B|3C site, the corresponding residues [Gly-1 to Lys-12 in human polio virus (HPV) 3C] must be in an extended conformation (Fig. 5I) to reach into the active site through the cleft between ß-strand bI from the N-terminal domain and the loop connecting ß-strands aII and bII from the C-terminal domain. The loop that connects ß-strands aII and bII had to be moved in the model of the precursor molecule (Fig. 5I), with respect to its position in the native HPV 3C protease structure (Fig. 5III) to accommodate this. Several residues from ß-strand aI also are slightly moved away from their positions in the structure of the native 3C protease to widen the cleft between the N- and C-terminal domains through which the N terminus passes.
After the autocatalytic cleavage at Gly-1 of HPV 3C the new N terminus dissociates out of the active site (Fig. 5II). The folding of residues 5–13 into a stable helix, which packs tightly onto the surface of the molecule, subsequently would render this conformational change irreversible. It is necessary to remove the new N terminus from the protease active site to prevent intramolecular, competitive product inhibition of the protease.
The conserved sequence motif K/RR/KNI/L that eventually forms the last turn of the N-terminal helix anchors the residues of the N-terminal helix to the core structure of the protease. The side chains of Arg-13 and Asn-14 interact with the highly conserved sequence motif KFRDI of the RNA-binding site of the 3C protease. The residues that will become the N-terminal helix are in an extended conformation in the precursor (Fig. 5I). The up-down side-chain pattern in this extended conformation places the small side chains of Ala-7, Ala-9, and Ala-11 (P7', P9', and P11') into the cleft between the two domains of the proteases and the larger side chains of residues Tyr-6, Val-8, and Met-10 point to the surface. Larger side chains than alanine in positions 7, 9, and 11 would not have fitted easily into the surface of the cleft. We suggest therefore that the three alanine residues are important for the conformation of the N terminus in the precursor as well as for the formation of the N-terminal helix.
It is much more difficult to envision an intramolecular cleavage of the picornaviral 3C protease at its own C terminus. The crystal structure of the core proteins from Sindbis and Semlicki forest viruses (66) show how an additional ß-strand can reach from the C terminus to the active site of a chymotrypsin-like protease; however, the unique antiparallel ß-ribbon of the picornaviral 3C proteases that extends from ß-strands bII and cII and interacts with the N-terminal domain would prevent this (Fig. 5III).
We thank Perry d’Obrennan for help in making Fig. 2. Mae Wylie has been very helpful in getting the manuscript into its final polished form. A.R.K. was supported by a Medical Research Council of Canada Studentship; N.K.-B. was the holder of an Alberta Heritage Foundation for Medical Research Studentship. This work has been supported by the Medical Research Council of Canada and by Grant UO1AI38249 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health.
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