Contribution of NA to virion binding signal.

(A-B) NA activity-dependent virus self-elution was examined for the first seconds (for PR8MtSIN; panel A) or minutes (for WSNHAMtSIN; panel B) of the dissociation phase. Viruses were loaded at 100 pM concentration to 3’SLN (PR8MtSIN) or 3’N Fetuin (WSNHAMtSIN) for 30 minutes in presence of 10 μM OC after which the sensors were washed in PBS and dissociation was examined at a range of OC concentrations as indicated in the panels. (C-F) PR8MtSIN (C, E) and WSNHAMtSIN (D, F), carrying the same HAMtSIN but either NAMtSIN (high NA activity) or NAWSN (low NA activity), respectively, were bound at 100 pM concentration to biotinylated 3’SLN (C, D) or Fc-tagged 3’N Fetuin (E, F) loaded to maximum level. Binding was performed in absence (red lines) or in the presence of a range of OC concentrations as indicated in the panels. Significant differences between initial binding curves shown in panels C-F were analyzed by IBM SPSS statistic 24. In panel C, there was no significant difference between curves with 10μM OC and 625nM OC (P>0.1), whereas the curves of 10μM OC and 625nM OC were significantly different from 39nM, 2.45nM and 0nM (P<0.001). In panel D, the curves of 10μM OC and 625nM were significantly different from those of 39nM, 2.45nM and 0nM. There also was a significant difference between 39nM and 2.45nM. In panel E, the 100nM curves significantly differed from the other three OC concentrations. In panel F, there were significant differences between the 100nM (and 25nM) and 6.25nM and 0nM curves. (G) The ratio (initial virus binding rate vobs in absence of OC)/(initial virus binding rate vobs in presence of 10 μM OC) of four viruses carrying the WSNWT NA in the background four different HAs was plotted (the individual binding curves are shown in S9 Fig). Standard deviations and significant differences between the mean initial binding rate ratios are indicated (* indicates P<0.05).