Table 2. Enzymatic activities and thermo-stabilities of DENV4 WT and mutant NS5 proteins.

% NS5 activity	De novo initiation/elongation			Elongation			N-7 MTase	2’-O MTase	Thermo-fluorescence
Time (hr)	1	2	3	1	2	3	0.25	1	Tm (°C)
WT	100	100	100	100	100	100	100	100	37
K95A	189.9 ± 4.0	198.4 ± 9.5	171.9 ± 3.7	194.0± 3.9	200.1 ± 1.0	182.3 ± 13.1	85.6 ± 15.0	82.4 ± 1.0	37
Y119A	149.5 ± 11.4	144.3 ± 22.1	149.9 ± 12.1	113.0 ± 3.3	110.2 ± 1.1	114.5 ± 0.5	46.3 ± 0.8	8.3 ± 0.4	38
R263A	151.3 ± 2.5	142.8 ± 4.9	135.7 ± 5.0	103.9 ± 4.8	105.0 ± 0.5	112.4 ± 0.8	4.0 ± 0.3	3.2 ± 0.3	37
E268A	132.8 ± 12.1*	123.9 ± 1.3*	146.4 ± 1.7*	92.7 ± 2.5	87.6 ± 2.8	98.3 ± 3.2	71.9 ± 2.6	70.4 ± 2.5	37
E270A	47.2 ± 6.4*	47.4 ± 5.6*	54.6 ± 10.2*	71.7 ±2.4	70.1 ± 1.8	77.9 ± 1.5	74.1 ± 10.2	65.1 ± 0.4	37
R353A	107.0 ± 3.1	92.0 ± 3.4	94.3 ± 2.5	193.1 ± 2.9	198.1 ± 25.1	206.4 ± 15.0	106.3 ± 6.7	97.7 ± 4.4	38

Polymerase activities DENV4 WT and mutant NS5 (full length) proteins measured in de novo initiation and elongation FAPA assays. Results shown are the average percentage activity compared against DENV4 WT NS5 protein derived from average relative fluorescence units (RFU) obtained for each protein from one experiment. All data points were performed in duplicate wells. N7 and 2’-O MTase of DENV4 WT and mutant NS5 proteins measured in SPA assays. Results shown are the average percentage activity compared against DENV4 WT NS5 protein derived from average corrected counts per minute (CCPM) obtained for each protein. Thermo-stability was assessed using the thermo-denaturation assay.

Footnote:

* Data source [32]. The effect of alanine mutations of E268 (E267 in DENV3), and E270(E269 in DENV3), on de novo initiation/elongation activities of polymerase were previously studied in Lim et al., 2013 and is included here for comparison.