Human rhinoviruses (HRVs)
Human rhinoviruses (HRVs) are small, non-enveloped viruses belonging to the genus Enterovirus of the family Small RNA Viruses, with three main species, HRV-A, HRV-B, and HRV-C, each of which has multiple serotypes. Human rhinovirus has a single-stranded RNA of about 7000bp, and this open reading frame encodes a polyprotein during viral infection, which is then split into three precursor polyproteins, P1, P2 and P3. Among them, the precursor polyprotein Pl is further cleaved into VP1, VP2, VP3 and VP4, and a single copy of these four structural proteins can form a viral progenitor, and then 60 progenitors can form an icosahedral capsid; while the precursor polyproteins P2 and P3 are further cleaved into seven non-structural proteins, of which 3C protease is one.
3C protease
Human Rhionvirus 3C Protease (HRV 3C protease), as a non-structural protein, is not involved in the assembly of the viral capsid but is involved in the cleavage and separation of most of the precursor proteins and is a key functional protein for the production of mature viral proteins and viral replication. Currently, only three crystal structures of human rhinovirus 3C protease are reported in the article, namely the 3C protease of serotypes HRV2, HRV14 and HRV16.
Structure
Although the substrate binding pocket of the various serotype human rhinovirus 3C proteases is highly conserved, there is a high degree of variability in the S2 active pocket. the 3C protease of HRV2 has non-conserved amino acid residues of Lys69 and Asn130 in the S2 active pocket, which are replaced with the medium volume amino acids Lys69 and Thr130 in the 3C protease of HRV16, whereas the 3C protease S2 activity pocket of HRV14 is composed of smaller volume amino acids Asn69 and Thr130, forming a larger S2 activity pocket. The larger S2 active pocket can accommodate a bulkier P2 substituent, and the protease-inhibitor interaction of the S2 pocket has a significant effect on the inhibitor activity. Thus, we can omit the molecular design of S3 and S4 pockets and directly obtain an inhibitor 2 with strong antiviral activity.
Inhibitors
For those who are susceptible to HRVs, infection with HRVs brings great distress to patients and their families, and great economic loss to society. Because RNA viruses do not possess a DNA repair system, RNA viruses are highly mutagenic and diverse. To avoid delays in drug development for new mutant viruses, the development of broad-spectrum inhibitors is a major research direction. In view of the key role of human rhinovirus 3C protease in virus proliferation, scientific research made to look at the king to design a series of inhibitors for the specificity of the protease S2, S1, S1' pocket. Among them, there are both screened and modified non-peptide reversible inhibitors, such as heterocyclic lipids1, as well as irreversible inhibitors of peptides.
Substrate cleavage sequence of HRV3C
The polyprotein encoded by human rhinovirus can be cleaved into 11 mature viral proteins and functional viral enzymes by cleavage, of which 8 sites are cleaved by human rhinovirus 3C protease. Based on these cleavage sites, we can know that the human rhinovirus 3C protease can specifically recognize a segment of amino acid sequence, and the cleavage reaction occurs between GIn-Gly, Gln-Ala and Glu-Gly. The results of one study showed that the human rhinovirus 3C protease showed maximum cleavage efficiency when it recognized the sequence DSLETLFQ↓GPVYKDLE between 2C and 3A. Later studies examined that the shortest amino acid recognition sequence of the human rhinovirus 3C protease is 6 amino acids (TLFO↓Gp) and shows high specificity against GIn and Gly at the PI and P1' sites, respectively. However, detailed amino acid specificity for these sites is still lacking.
