Fig. 6.

Dissociation of gL from virion gH/gL at infection of human epithelial SW480 cell line. β6- or β8-integrins were silenced singly (siRNA β6 or siRNA β8), doubly (siRNA β6+β8), or mock-silenced (siRNA ctrl) in SW480 cells. HSV-1 was absorbed to the integrin-depleted cells for 90 min; virus absorption and dissociation of gL were quantified as flow cytometry reactivity to MAbs 52S (red) and 53S (blue), respectively. (A–C) Extent of silencing was measured in uninfected cells by qRT-PCR (A) and expressed as fold decrease relative to siRNA ctrl cells or by flow cytometry (B and C). In A, each column represents the average of triplicates from two independent experiments ± SD. ***P < 0.001. (D–G) Flow cytometry reactivity of cells carrying absorbed virions to MAbs 53S (blue) and 52S (red) was expressed as the percentage of positive cells (figures within each panel). (H) The 53S (blue) reactivity shown in D–G was expressed as a percentage of the 52S (red) reactivity. (I) At the end of virus absorption, the media from the SW480 cells shown in D–G were analyzed for the presence of released gL, as detailed in Fig. 4C. It can be seen that gL was released from virions absorbed to siRNA ctrl-treated SW480 cells, was released in a small amount from virions absorbed to singly silenced SW480 cells, and was undetectable in media of SW480 cells simultaneously silenced for both integrins. Lane HSV shows the WB immunoreactivity of the indicated virion glycoproteins.