FIG 3 .

Lineage C NS4b proteins are catalytically active 2′,5′-phosphodiesterases. (A) Abrogation of catalytic activity by mutation of the second catalytic His of NS4b proteins and comparison with MHV NS2 and RVA VP3 CTD. The kinetic data were obtained using 10 µM 2-5A and 1 µM enzyme concentration with the exception of MBP, which was used at 10 µM. Data shown are from one representative experiment of three carried out with separate enzyme preparations. (B to D) Steady-state enzyme kinetic curves of velocity versus concentration of 2-5A for MHV NS2 (B), MERS-CoV (C), BtCov-SC2013 (D), and BtCoV-HKU5 (E) NS4b proteins. Kinetic reactions were carried out in triplicate and expressed as means ± standard errors of the means (SEM) (error bars). Substrate-dependent velocity measurements are shown from one representative experiment carried out twice in triplicate with separate enzyme preparations.