Biophysical characterization of HCV Q65H NS5B and eIF4B: Protein-nucleic acid interactions

Hepatitis C Virus (HCV) infection is one of the most common chronic blood borne infections. The mechanism underlying persistence of HCV infections are not well understood. Mutation of Gln65His (Q65H), which develops in the NS5B dependent RNA polymerase (RdRp) during chronic infection, has 1.8 fold enhanced invitro RdRp activity, may have major implications in the efficiency of HCV RNA replication and viral persistence. The reason for this increase in activity is not well understood (Virology 2003 Dec 5; 317(1):65-72). We used endogenous tryptophan fluorescence to characterize the interactions of Q65H protein with GTP, GDP, Mg2+ and a 19mer RNA template. The Q65H protein exhibits 3 fold lower Kd (11.6 +/- 1.2 muM) than wildtype (36.8 +/- 1.0 muM) for GTP, whereas the Kd values of the mutant for MgCl2 and 19 mer RNA template were similar to wildtype protein. Thermodynamic studies on the binding reaction of the Q65H variant were also performed to provide details of the energetic and entropic characteristics.;The second part of this study involved interaction of eIF4B with zinc. eIF4B promotes RNA dependent ATP hydrolysis and ATP dependent RNA helicase activity of eIF4A and eIF4F. It has recently been reported that eIF4B also organizes assembly of the RNA, eIFiso4G, eIF4A, and poly-(A) binding protein and that zinc enhances the RNA binding and interaction with PABP. We previously reported that zinc binds tightly with eIF4B with a Kd of 19.7 +/- 1.6 nM (J Biol Chem. 2008 Dec 26; 283(52):36140--53). The Kd of binding of eIF4B with 20mer poly (A) RNA is 77 +/- 7 nM and in presence of zinc is reduced down to 45 +/- 3 nM. Circular dichroism showed that addition of zinc resulted in a more than 50% decrease in alpha content of the eIF4B protein. To better understand the role of eIF4B in the selection of RNA, we also studied the interaction of eIF4B with structurally different RNAs and the effect eIF4A on this interaction using fluorescence anisotropy. This overall study on eIF4B helps towards the better understanding of basic translation initiation machinery and some of its regulators.