Human rhinoviruses (HRVs), the major etiologic agents of the common cold in humans, contain over a hundred distinct serotypes and belong to the picornavirus family. HRV 2A protease cleaves the polyprotein in a cotranslational event.
Small plus-strand human rhinoviruses (HRVs), encode a single open reading frame which is translated into a single large polyprotein with a size of 220 kDa. Maturation cleavage of the polyprotein to generate functional viral proteins is mainly performed by two virally encoded proteases, designated 2A and 3C. The first cleavage is catalyzed by the 2A protease, which takes place at the junction of capsid protein VP1 and the N terminus of the 2A protease itself, separates the viral capsid from the nonstructural proteins. Most of the remaining cleavages are further processed by either the 3C protease or its precursor 3CD enzyme 1,2. Substrate requirements of the human rhinovirus serotype-2 2A protease have been examined using synthetic peptides 3. The natural variation in the amino acid sequences amongst rhinoviruses and enteroviruses at the cleavage site of the viral proteinase 2A served as the basis for a mutational analysis of the substrate specificity of the 2A proteinase of human rhinovirus 2 4. Homophthalimides are identified as potent inhibitors for rhinovirus protease, 2A. Several homophthalimides exhibit time-dependent inhibition of the 2A protease in the low-micromolar range, and enzyme-inhibitor complexes were identified by mass spectrometry 5.
The natural substrate for Human Rhinovirus-14 (HRV14) 2A Protease is the viral poly protein. The first cleavage of the polyprotein is catalyzed by the 2A protease as a cotranslational event. This cleavage, taking place at the junction of capsid protein VP1 and the N terminus of 2A itself, separates the viral capsid proteins from the nonstructural ones. In addition, 2A have been shown to be responsible for cleavage of several other important cellular proteins, which may lead to inhibition of normal host cell functions. 2A is an HRV-encoded cysteine protease and the small peptide substrates structural preference is for glycine and proline at the P1' and P2' positions, respectively. 2A also prefers several amino acids, including alanine, methionine, and tyrosine, at p1 position. Chromogenic octapeptide, R-K-G-D-I-K-S-Y-pNA, with its amino acid sequence derived from the authentic HRV14 2A cleavage site of the viral polyprotein, was synthesized and used as a substrate for HRV14 2A in vitro 2,5,6.
Mechanism of action
The HRV 2A protease has been proposed to be a zinc-binding protein. Sommergruber and his colleagues have demonstrated that zinc is essential for the structural integrity of the HRV2 2A protease. Competition cleavage assays reveal this peptide was cleaved over 10-fold more efficiently than the 16-mer peptide derived directly from its native processing site. Results suggest that P1' glycine residue is not absolutely needed for the 2A cleavage to occur and the essential residues required for the 2A activity would exist within the N-terminal side of the scissile bond 6,7. HRV2 2A only cleaved peptide substrates derived from other rhinovirus serotypes and poliovirus that possessed P2 Thr and P1' Gly. Thus, the sequence Thr-X-Gly may form the basis of the cellular cleavage site processed by rhinoviral 2As during viral replication. Compound LY343814, one of the most potent inhibitors against HRV14 2A protease, had an antiviral 50% inhibitory concentration of 4.2 µM in the cell-based assay. Results show that homophthalimides are not only 3C but also 2A protease inhibitors in vitro, implying that the antiviral activity associated with these compounds might result from inactivation of both 2A and 3C proteases in vivo. Since the processing of the viral polyprotein is hierarchical, dual inhibition of the two enzymes may result in cooperative inhibition of viral replication 5.
Antiviral drug development, the 2A proteases encoded by human rhinoviruses (HRVs) is attractive targets for antiviral drug development due to their important roles in viral replication.
IRES stimulations, The internal ribosome entry site (IRES) of enteroviruses, IRES activity can also be stimulated by the cleavage of initiation factor eIF4G mediated by either HRV 2A proteaseRhinovirus 2A proteinase mediated stimulation of rhinovirus RNA translation is additive to the stimulation effected by cellular RNA binding proteins 8.
Cytokeratin 8, a member of the intermediate filament group of proteins, was found to be proteolytically cleaved in vitro by the 2A proteinase of HRV2. Compared with proteolysis of the eIF4G homologues, K8 is cleaved late in the infection cycle 9.
1. Palmenberg AC (1990). Proteolytic procession of picornaviral polyprotein. Annu. Rev. Microbiol., 44: 603–623.
2. Porter AG (1993). Picornavirus nonstructural proteins: emerging roles in virus replication and inhibition of host cell functions. J. Virol., 67: 6917–6921.
3. Wang QM, Sommergruber W, Johnson RB (1997). Cleavage specificity of human rhinovirus-2 2A protease for peptide substrates. Biochem Biophys Res Commun., 235(3): 562-6.
4. Skern T, Sommergruber W, Auer H, Volkmann P, Zorn M, Liebig HD, Fessl F, Blaas D, Kuechler E (1991). Substrate requirements of a human rhinoviral 2A proteinase. Virology, 181(1): 46-54.
5. Wang QM et al., (1998). Dual Inhibition of Human Rhinovirus 2A and 3C Proteases by Homophthalimides. Antimicrobial Agents and Chemotherapy., 42(9): 916-920.
6. Sommergruber W et al., (1992). Cleavage specificity on synthetic peptide substrates of human rhinovirus 2 proteinase 2A. The Journal of Biological Chemistry, 267: 22639-22644.
7. Wang QM et al., (1998). Development ofin VitroPeptide Substrates for Human Rhinovirus-14 2A Protease*1. Archives of Biochemistry and Biophysics., 356(1): 12-18.
8. Hunt SL et al., (1999). Rhinovirus 2A proteinase mediated stimulation of rhinovirus RNA translation is additive to the stimulation effected by cellular RNA binding proteins. Virus Research, 62(2): 119-128.
9. Seipelt J et al., (2000). 2A Proteinase of Human Rhinovirus Cleaves Cytokeratin 8 in Infected HeLa Cells*. The Journal of Biological Chemistry, 275: 20084-20089.
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