Molecular cloning and sequence determination of the genomic regions encoding protease and genome-linked protein of three picornaviruses

Abstract

To investigate the degree of similarity between picornavirus proteases, we cloned the genomic cDNAs of an enterovirus, echovirus 9 (strain Barty), and two rhinoviruses, serotypes 1A and 14LP, and determined the nucleotide sequence of the region which, by analogy to poliovirus, encodes the protease. The nucleotide sequence of the region encoding the genome-linked protein VPg, immediately adjacent to the protease, was also determined. Comparison of nucleotide and deduced amino acid sequences with other available picornavirus sequences showed remarkable homology in proteases and among VPgs. Three highly conserved peptide regions were identified in the protease; one of these is specific for human picornaviruses and has no obvious counterpart in encephalomyocarditis virus, foot-and-mouth disease virus, or cowpea mosaic virus proteases. Within the other two peptide regions two conserved amino acids, Cys 147 and His 161, could be the reactive residues of the active site. We used a statistical method to predict certain features of the secondary structures, such as alpha helices, beta sheets, and turns, and found many of these conformations to be conserved. The hydropathy profiles of the compared proteases were also strikingly similar. Thus, the proteases of human picornaviruses very probably have a similar three-dimensional structure.

In Vitro Antiviral Activity of AG7088, a Potent Inhibitor of Human Rhinovirus 3C Protease

“…In cell-based assays, AG7088 demonstrated comparable an- (8,16,20,21,29,31,36,38,45,48,49,55). This same level of homology, reflected in the 3C protease-coding regions derived from other picornaviruses, is also consistent with levels of activity demonstrated against four related picornaviruses.…”

“…The HRV 3C protease is responsible for the cleavage of viral precursor polyproteins into structural and enzymatic proteins which are essential for viral replication. DNA sequence comparisons among HRV serotypes, and even among several related picornaviruses, have identified a significant degree of homology within the 3C coding region including the strict conservation of the active-site residues, thus providing an additional rationale for targeting drug discovery efforts (8,16,20,21,29,31,38,45,48,49,55). AG7088 is a potent, irreversible inhibitor of HRV 3C protease that was discovered by protein structure-based drug design methodologies (12,35,36) and is currently undergoing evaluation in phase I clinical studies with humans.…”

Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3

“…Butterworth and Korant (11) reported the observation of large picornaviral peptides in the presence of zinc ions, and Pelham (44) and Gorbalenya and Svitkin (21) reported a specific inhibition of 3C protease from EMC virus by NEM and iodoacetamide. On the basis of these data, and since picornaviral and plant comovirus 3C proteases are related cysteine proteases (3,19,61; for a review, see reference 32) with homology to cellular cysteine and serine proteases (20), we suggest that the FMDV 3C protease is involved in this specific histone H3-Pi transition.…”

“…In this mutant, amino acid residue Cys-163 was changed to a serine residue by sitedirected mutagenesis (Zibert and Beck, in preparation). By analogy to related cysteine proteases from other picornaviruses and comoviruses (3,19,61), Ser-163 is located in the active site of FMDV 3C protease. This protease mutant is also incapable of processing the viral polyprotein in vitro (data not shown).…”

Molecular Typing of Enteroviruses: Current Status and Future Requirements

“…Complete genome sequences for a number of human enterovirus serotypes have been published (51,60,92,139,173,174,179,182,190,217,221,232,246,317,337,339,353,384,413,414,419,431,487,493) (Table 2). Partial sequence data are available for the majority of the remaining serotypes (23,103,105,130,177,339,342,349,372,461) (Table 2). While sequence comparisons partially support the classical subgrouping of enterovirus serotypes into PV, CAV, CBV, and ECV, in many cases genetic relationships do not correlate with this division.…”