title: Development of robust in vitro serine protease assay based on recombinant Pakistani HCV NS3-4A protease.
abstract: Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 3 (predominant genotype in Pakistan) and they are poorly tolerated, highlighting the requirement of new therapeutics. HCV non-structural protein-3 (NS3) protease and helicase domains are essential for viral replication; they are highly conserved among various HCV strains. In the current study, we enrolled 56 HCV infected patients from various regions of Pakistan and determined their genotypes, ALT level and virus titer. We have cloned and sequenced NS3/NS4A from 4 of the HCV Serum samples. Nucleotide sequence alignment showed high level of identities among 3a genotypes. One of the samples (NCVI 01) showed unique amino acids substitutions, including R9Q, L332P, L354I, I605V and S622C. Three dimensional structures were determined and analyzed effect of substitutions on amino acids interactions. We further established fluorescence resonance energy transfer (FRET) based assays for detecting proteolytic activity of (NS3-4A) serine protease, using AnaSpec peptide, for high throughput screening (HTS) inhibitors against HCV. In future, this study could be of great interest in the development of HCV NS3 cell-based HTS FRET assay for genotype 3a and subsequent antiviral testing of drugs.
authors.affiliation: NUST Center of Virology and Immunology, National University of Sciences and Technology, Sector H-12, Islamabad, Pakistan.
authors.forename: Kaneez
authors.initials: K
authors.lastname: Fatima
authors.forename: Muhammad
authors.initials: M
authors.lastname: Tahir
authors.forename: Ishtiaq
authors.initials: I
authors.lastname: Qadri
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000821:virology; D057166:High-Throughput Screening Assays; D006801:Humans; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010154:Pakistan; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D012367:RNA, Viral / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055815:Young Adult; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus