Figure 6.
SV5 displays reduced binding but increased spread compared with T3wt in L929 cells
(A) Binding of T3wt, SV5, and single mutated viruses on L929 cells. Percentage of binding was calculated by western blot analysis comparing the amount of viral proteins bound to the cells at 0 hpi versus the input for each virus (n = 3). ∗p < 0.05; ns, not significant (ANOVA with Tukey test). (B) Representative agarose gel showing σ1 levels in intact virions. 1% agarose gels were loaded with the same number of virus particles. Band migration corresponds to σ1 trimer levels per virion (n = 3).52 (C) Graph showing the amount of virions presenting determined σ1 levels (n = 3). (D and F) Single-step growth curves showing cellular, total, and released titers (n = 2). L929 cells were infected at an MOI that infected more than 70% of the cells (checked by flow cytometry). Cellular lysates and supernatants (released titers) were collected at 0, 3, 6, 9 12, 15, 18, 24, 30, and 36 hpi and titered in L929 cells. Total titers were calculated as the sum of cellular and released titers. (G) Cell death evaluated by Zombie Aqua staining (by flow cytometry). Top: cell death evaluated at 24 hpi when L929 cells were infected at MOIs of 3 and 27 PFU/cell of T3wt or SV5. Bottom: cell death measured at different times of infection (15, 18, 24, 30, and 36 hpi) when L929 cells were infected at an MOI of 27 PFU/cell of T3wt or SV5. (H) Representative immunofluorescence images of T3wt and SV5 plaque formation at days 2, 3, and 4 post-infection (n = 3). In pink is anti-reovirus staining (rabbit polyclonal anti-reovirus), in green the σNS staining (non-structural reovirus protein, 2A9 mouse monoclonal antibody), in yellow the colocalization of both colors, and in blue the nuclei. Scale bars, 500 μm. (I) Quantification of the fluorescence from the edge of the circular plaque outward into the neighboring monolayer of at least 50 plaques per day post infection. Each separate line represents the average of the fluorescence presence from the edge of the plaque per plaque. Thick colored lines were calculated using a local polynomial regression fitting formula (LOESS).