Characterization of NS3 protease from an Egyptian HCV genotype 4a isolate.
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.
Bahgat
Mahmoud Mohamed
MM
The Immunology and Infectious Diseases Laboratory, Therapeutic Chemistry Department, The Center of Excellence for Advanced Sciences, The National Research Center, Cairo, Egypt. mbahgatriad@yahoo.com
Ibrahim
Ahmed Atef
AA
Abd-Elshafy
Dina Nadeem
DN
Mesalam
Ahmed Atef
AA
Gewaid
Hossam Eid
HE
Ismaeil
Amany Abd-Elghany
AA
El-Waseef
Ahmed Mohamed
AM
Maghraby
Amany Sayed
AS
Barakat
Ahmed Barakat
AB
El-Far
Mohamed Abd-Elhafez
MA
Ghanem
Hossam El-Din Ahmed
Hel-D
Mohamed
Amro Mahmoud
AM
Ali
Mohamed Ahmed
MA
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004534:Egypt; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005838:Genotype; D016174:Hepacivirus / Q000201:enzymology* / Q000235:genetics; D006526:Hepatitis C / Q000821:virology; D006801:Humans; D008264:Macrophages / Q000821:virology; D051379:Mice; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics / Q000502:physiology; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases