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.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Marohnic CC,Birkenmeyer LG,Bogdan MF,Frias EC,Otis KS,Palafox MAP,McSherry TD,Gregory SD,Zhao C,Gutierrez RA,Prostko JC,Muerhoff ASdoi
10.1016/j.jviromet.2018.10.019subject
Has Abstractpub_date
2019-02-01 00:00:00pages
11-17eissn
0166-0934issn
1879-0984pii
S0166-0934(18)30398-7journal_volume
264pub_type
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