Engineering of the hepatitis C virus helicase for enhanced seroreactivity.
J Virol Methods
; 264: 11-17, 2019 02.
Article
em En
| MEDLINE
| ID: mdl-30381240
ABSTRACT
Hepatitis C Virus c33, a recombinant protein comprising residues 1192-1457 of NS3 helicase, has been a mainstay of HCV serology for decades. With seven unpaired cysteines, seroreactivity of E. coli expressed c33 is dependant on reductants. While engineering a c33 replacement for new anti-HCV serological tests, we sought to reduce oxidation sensitivity, a liability for immunodiagnostic reagent stability. A series of cysteine-to-serine substituted variants of a c33-like antigen was constructed and evaluated for reactivity against a panel of HCV-positive sera. Several variants were essentially nonreactive while others exhibited reactivity similar to or better than the wild-type construct. One demonstrated equivalent potency to wild-type but also diminished DTT dependence. To explore enhanced anti-NS3 reactivity, we constructed and examined an expanded series of antigens comprising individual helicase domains, the full-length helicase, additional cysteine-to-serine variants, and variants at positions critical to catalytic activity. Immunoassays using these latter NS3 helicase recombinants demonstrated that domain 1 possessed significantly more seroreactivity than previously believed, that the use of soluble full-length helicase protein enhanced sensitivity by several-fold over c33, and that anti-NS3 helicase seroreactivity was further enhanced by the introduction of point mutations which altered the catalytic activity or oxidation sensitivity of the antigen.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Testes Sorológicos
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Proteínas não Estruturais Virais
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DNA Helicases
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Hepacivirus
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article