Alexei P. Donchenko

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold.

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin‐like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como‐, nepo‐ and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re‐identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C‐terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N‐terminal, and Cys in the C‐terminal β‐barrel domain. These residues are suggested to form a charge‐transfer system similar to that formed by the catalytic triad of chymotrypsin‐like proteases. Based on the structural analogy with chymotrypsin‐like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate‐binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.

A novel superfamily of nucleoside triphosphate-binding motif containing proteins which are probably involved in duplex unwinding in DNA and RNA replication and recombination.

A statistically significant similarity was demonstrated between the amino acid sequences of 4 Escherichia coli helicases and helicase subunits, a family of non‐structural proteins of eukaryotic positive‐strand RNA viruses and 2 herpesvirus proteins all of which contain an NTP‐binding sequence motif. Based on sequence analysis and secondary structure predictions, a generalized structural model for the ATP‐binding core is proposed. It is suggested that all these proteins constitute a superfamily of helicases (or helicase subunits) involved in NTP‐dependent duplex unwinding during DNA and RNA replication and recombination.

An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication.

SummaryNTP-motif, a consensus sequence previously shown to be characteristic of numerous NTP-utilizing enzymes, was identified in nonstructural proteins of several groups of positive-strand RNA viruses. These groups include picorna-, alpha-, and coronaviruses infecting animals and como-, poty-, tobamo-, tricorna-, hordei-, and furoviruses of plants, totalling 21 viruses. It has been demonstrated that the viral NTP-motif-containing proteins constitute three distinct families, the sequences within each family being similar to each other at a statistically highly significant level. A lower, but still valid similarity has also been revealed between the families. An overall alignment has been generated, which includes several highly conserved sequence stretches. The two most prominent of the latter contain the socalled “A” and “B” sites of the NTP-motif, with four of the five invariant amino acid residues observed within these sequences. These observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the NTP-motif-containing proteins of positive-strand RNA viruses are homologous, constituting a highly diverged monophyletic group. In this group the “A” and “B” sites of the NTP-motif are the most conserved sequences and, by inference, should play the principal role in the functioning of the proteins. A hypothesis is proposed that all these proteins posses NTP-binding capacity and possibly NTPase activity, performing some NTP-dependent function in viral RNA replication. The importance of phylogenetic analysis for the assessment of the significance of the occurrence of the NTP-motif (and of sequence motifs of this sort in general) in proteins is emphasized.

Poliovirus-encoded proteinase 3C: a possible evolutionary link between cellular serine and cysteine proteinase families

Here we demonstrate significant similarities between the amino acid sequences of trypsin (a serine protease) and the N‐terminal piece of a specific fragment of the poliovirus polyprotein encompassing the sequence of the viral proteinase 3C, and also between cathepsin H (a cysteine protease) and the C‐terminal piece of the same fragment. A coherent alignment of the sequences of the 3 proteases was obtained, in which the principal catalytically active residues occupy identical positions. A hypothesis is proposed that the viral enzyme may provide an evolutionary link between serine and cysteine protease families.

Sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses.

A putative serine protease was identified among non‐structural proteins of southern bean mosaic virus (SBMV) by sequence comparison with cellular and viral proteases. The predicted SBMV proteased is played a significant similarity to cysteine proteases of picornaviruses, providing a possible evolutionary link between the two enzyme classes. It is suggested that SBMV follows the general expression strategy characteristic of other positive‐strand RNA viruses containing 5′‐terminal covalently linked proteins (VPg), i.e. generation of functional proteins by polyprotein processing.