Skip to main content
. 2012 Nov;86(22):12148–12160. doi: 10.1128/JVI.01133-12

Fig 8.

Fig 8

HMPV fusion is not triggered by HMPV G or RGD-binding integrins. (A) R18 F-VLP fusion was measured in the absence (circles) or presence (triangles) of neutralizing HMPV antiserum or for heat-inactivated particles (squares). R18-G-VLP (diamonds) dequenching was monitored as a background control. Percent R18 dequenching was calculated as described in Materials and Methods, and curves represent means for three independent experiments monitored for triplicate wells. Error bars indicate SEM. (B and C) HMPV G does not alter HMPV F-mediated fusion kinetics or the extent of fusion at 4 h. R18-labeled HMPV (black circles), F-VLPs (gray squares), or F+G-VLPs (open squares) were bound to the surface of BEAS-2B cells, and R18 fluorescence was monitored for 4 h. Triton X-100 was added after 4 h to determine the extent of virus or VLP fusion. Curves in panel B represent mean percent R18 dequenching for three independent experiments, monitored for duplicate wells. Bars in panel C represent the extent of fusion observed after 4 h for three independent experiments; error bars indicate SEM. *, P < 0.05; N.S., P > 0.05. (D to F) HMPV F binding to RGD-binding integrins does not alter HMPV fusion kinetics. R18-labeled HMPV (D), F-VLP (E), or F+G-VLP (F) fusion was assessed in the absence of antibodies (black lines) or in the presence of integrin function-blocking antibodies against α2 integrins (gray lines) or all RGD-binding integrins (αV plus α5 plus β1) (dotted lines). Curves represent mean percent R18 dequenching for three independent experiments monitored for duplicate wells. Error bars are not shown for figure clarity. Dequenching rates were not significantly altered in the presence of integrin antibodies; however, significantly less HMPV and VLPs bound during RGD-binding integrin blockade (data not shown). (G) F-RGD (wt) VLPs (10 μg), F-RAE VLPs (10 μg), and producer cell lysates (50 μg) were analyzed by Western blotting. Uncleaved (F0) and cleaved (F1) HMPV F were detected with an F-specific MAb and fluorescent secondary antibody using the Li-Cor Odyssey infrared imaging system. C, untransfected 293-F cell lysate. (H) The RGD integrin-binding motif is not required for efficient F-mediated hemifusion. R18 VLP fusion was measured for F-RGD (circles), F-RAE (triangles), or G-only (squares) particles as described for panel A.