The human rhinoviruses (HRVs), implicated as the major causative agents of upper respiratory tract infections collectively known as the common cold, belong to the largest genus of the picornavirus family. 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.

Structural Characteristics
From a structural point of view, rhinovirus 3C proteins display a strong similarity to trypsin-like serine proteases, 3C contains a cysteine as the active site nucleophile. The X-ray crystal structures of 3C proteases from both hepatitis A virus and HRV14 have been solved, revealing their structural similarity to the typical serine proteases 3. Colorimetric assay for 3C using peptide p-nitroanilides (pNA) as substrates peptides suggests amino acids downstream from the original cleavage site have all been replaced with a chromophoric p-nitroaniline moiety which is directly linked to the bond undergoing enzymatic cleavage, thereby generating a new cleavage site Gln-pNA for the enzyme 4.Cleavage by the 3C proteinase are predicted to occur at a Gln-Gly junction. The hydrolysis was shown (by reverse phase fast protein liquid chromatography and amino acid analysis) to occur specifically at the Gln-Gly bond in each of the peptides. The ready availability of such convenient substrates facilitated the further characterization of the 3C proteinase 5.

Mode of Action
The ability of the HRV-14 3C proteinase to hydrolyse the synthetic peptides was inhibited if a Cys~Ser(146) mutation was introduced into the protein. Studies with known proteinase inhibitors substantiated the conclusion that the HRV- 14 3C protein appears to be a cysteine proteinase and that the Cys residue at position 146 may be the active site nucleophile 5. Kinetic parameters of 3C protease toward p-nitroanilides (pNA) peptides have been measured and analyzed. The pNA peptides have been modeled within the active site of the 3C protease to investigate the ability of the pNA group to act as a replacement for Gly-Pro in the prime side. Hydrolysis of these pNA peptides by 3C at the newly formed scissile bond releases free p-nitroaniline which is yellow-colored and can be continuously monitored at a visible wavelength 3. Compound LY343814, one of the most potent inhibitors against HRV14 3C protease, had an antiviral 50% inhibitory concentration of 4.2 µM in the cell-based assay. Results suggest 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 4.


Drug development, the 3C proteases encoded by HRV are attractive targets for antiviral drug development due to their important roles in viral replication. The HRV-14 3C proteinase probably plays animportant role, analogous to that implied for the poliovirus 3C proteinase, in the replication of the virus and thus represents a potential target for antiviral chemotherapy 6.

RNA binding, in addition to its proteolytic activity, viral 3C protease has been shown to be a RNA-binding protein and may be involved in formation of the viral replication complex .

Serine protease, as illustrated by its crystal structure, HRV 3C protease represents a novel class of cysteine protease that contains a cysteine as the active site nucleophile but is structurally like a serine protease 7.


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.     Malcolm BA (1995). The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds. Protein Sci., 4:1439–1445.

4.     Wang QM, Johnson RB, Cox GA, Villarreal EC, Loncharich RJ (1997). A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates. Anal Biochem., 252(2):238-245.

5.     David C,  LONG AC,  Kay J, Dunn BM, Cameron  JM (1989). Hydrolysis of a Series S of synthetic Peptide Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned and Expressed in E coli. J. gen. Virol., 70: 2931-2942.

6.     Leong LE, Walker PA, Porter AG (1993). Human rhinovirus-14 protease 3C (3Cpro) binds specifically to the 5'-noncoding region of the viral RNA. Evidence that 3Cpro has different domains for the RNA binding and proteolytic activities. J. Biol. Chem., 268: 25735–25739.

7.     Bazan JF, Fletterick RJ (1988). Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.PNAS., 85:7872–7876.

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