Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization

The Biochemical Journal
Hannelore PetersVerena Gauss-Müller

Abstract

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24-->Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (K(D)) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by k(on) and k(off) values of the order of 10(2) M(-1) x s(-1) and 10(-2) to 10(-1) s(-1) respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5'-terminal domain, followed fast binding kinetics with k(on) and k(off) values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Wh...Continue Reading

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Citations

Dec 14, 2011·Nucleic Acids Research·Bärbel S BlaumGeorg Sczakiel
Apr 28, 2006·Journal of Virology·Corinne E ZeitlerB V Venkataram Prasad
Mar 15, 2011·Journal of Molecular Biology·Sheng CuiQi Jin
Feb 8, 2008·BioTechniques·Andrey L MikheikinAlexander S Zasedatelev
Nov 28, 2006·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Jan 11, 2019·Journal of Virology·Mariya A ViskovskaB V Venkataram Prasad
Oct 20, 2010·Magnetic Resonance in Chemistry : MRC·Youlin XiaXiaolian Gao

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