Fig. 5: The K4N mutation in E2 improved the attachment and entry of M1 virus.

a A modified plaque formation assay was performed and cells were imaged with a fluorescence microscope 48 hours after infection (MOI = 0.5). Scale bars, 500 μm. The scale bars in the magnified images represent 100 μm. b Plaques were counted with a fluorescence microscope 48 hours after infection. Data points represent mean plaque number per well ± SD, for n  =  3 biological replicates. P  <  0.0001 was determined by Two-way ANOVA relative to M1-GFP. c HCT-116 cells were incubated with M1-GFP and M1-E2M at 4 °C for 1 hours. Viral RNA was quantified by qRT-PCR and presented as mean ± SD, for n  =  3 biological replicates. P  <  0.0001 was calculated with Two-tailed unpaired t-test. d Western blot analysis of M1-GFP and M1-E2M incubated with MXRA8-His bound to His-Tag Mouse mAb Sepharose Beads. Precipitated viral particles were detected using an anti-E1 mAb (left). Quantification of E1 expression is shown (right). Graph bars represent mean densitometry of E1 normalized to the densitometry of MXRA8 ± SD, for n  =  3 biological replicates. P  = 0.0060 was calculated with Two-tailed unpaired t-test. e Time course of the binding between M1 viral particles and the MXRA8 protein, as determined via BLI. f, g HeLa, HeLa-Mxra8, Hs 578 T and Hs 578T-ΔMxra8 cells were treated with M1-GFP or M1-E2M. The infection rate was determined by flow cytometry 48 hours after infection. Graph bars represent mean infection rate % ± SD, for n  =  3 biological replicates. Statistical significance was calculated using Two-way ANOVA with Sidak’s multiple comparisons test relative to M1-GFP. Adjusted P values are: f vector P = 0.6199; Mxra8 P = 0.0002; g vector, P = 0.0010; ΔMxra8 P = 0.2713. n.s.: no significance, **P  <  0.01, ***P  <  0.001, ****P  <  0.0001. Source data are provided as a Source Data file.