Characterization of NS3 protease from an Egyptian HCV genotype 4a isolate

Abstract

The role of the NS3 protease in HCV replication was demonstrated by the ability of a protease inhibitor cocktail (10 microg/ml) to abolish the induced cytopathic effect in RAW macrophages upon infection with Egyptian sera. The HCV protease gene was amplified from Egyptian sera by nested PCR and cloned downstream of the CMV promotor in a mammalian expression plasmid, which was then used to transform bacteria. Colonies carrying the gene in the correct orientation were subjected to large-scale plasmid purification followed by sequencing. Phylogenetic comparison of the sequence obtained with published sequences from different genotypes confirmed that our sequence belongs to genotype 4a. Of the other genotypes, the most closely related ones were from genotype 1. Multiple alignments of protease peptides showed that the catalytic triads and binding residues for substrate, Zn2+ and the NS4 cofactor are conserved among different isolates, including ours, and confirmed the closer homology between NS3 of genotypes 4 and 1. The HCV-protease-encoding construct was successfully transcribed in both mammalian cells and mice. Mouse antibodies produced against the protease-encoding-construct detected the 18-kDa enzyme in lysates of cells transfected with the construct by Western blotting, and in the media of infected cells by ELISA.

Ligand design, synthesis and biological anti-HCV evaluations for genotypes 1b and 4a of certain 4-(3- & 4-[3-(3,5-dibromo-4-hydroxyphenyl)-propylamino]phenyl) butyric acids and 3-(3,5-dibromo-4-hydroxyphenyl)-propylamino-acetamidobenzoic acid esters

“…Thus, these compounds (Xa and Xb) would be reported here, as the first time, as prototypes for defeating genotype 4a of HCV infections. Such results were consistent with their docking scores at the 3D structure of binding site of the HCV NS3 protease enzyme that is conserved in both genotypes 1b and 4a [21]. In the other hand,…”

“…Such enzyme was reported to be the most important selective drug target for potential anti-HCV agents, where various inhibitors for this target were developed and are now in clinical trials [19] (Supplementary materials). The importance of this target are i) It is well-characterized HCV enzymes [20] and have a conserved binding site among all the HCV genotypes [21], ii) it has a successful use in HIV/AIDS therapeutics [22] and iii) It restores the host cell's antiviral defense [23].…”

“…Actually, the 3D crystal structure of HCV protease of genotype 1b is successfully isolated and is available in the www.pdb.org web in many codes including; 1W3C, while 3D crystal structure of the genotype 4a is not recognized up till now and hence it is not available in pdb web. However, this problem was solved by Bahgat et al finding [21], for the genomic sequence heterogeneity of the HCV proteases enzymes from different isolates (including genotypes 1b and 4a). The same authors demonstrated that the protease enzyme of the genotype 4a has closer homology to that of the genotype 1b.…”

Interaction Between the Neglected Tropical Disease Human Schistosomiasis and HCV Infection in Egypt: a Puzzling Relationship

“… 99 Altogether, these data suggest that HCV- NS3/4A protease is an attractive anti-HCV drug target, and therefore, I and others molecularly characterized this protease and reported on selective serine protease inhibitors as potential anti-HCV therapeutics. 90 , 100 – 103 …”

Schistosoma mansoni soluble egg antigens enhance HCV replication in mammalian cells

“…Uncoated spaces on plate wells were blocked against non specific binding by being incubated with PBS-0.05% Tween-5%FBS (PBST-FBS; 200 µl/well) at 37°C for 2 hours. After 3 washes, wells were incubated with 100 µl of the first antibody which was either anti-HCV antibody positive human sera or mouse anti-sera raised against a DNA mammalian expression construct encoding HCV-NS3 protease [29] or mouse anti-HCV core monoclonal antibody (Virogen, Watertown, MA, USA) at 37°C for 2 hours. Both human sera or mice anti-NS3 antibodies were used at a dilution of 1:100 while the mouse anti-core-antibody was used at 1:2000 in PBST-FBS.…”