Fig. 2: Knocking out Rab27a restricts IAV assembly and budding.

a Rab27a-KO and A549-Cas9 cells were infected with HM virus (MOI, 10). At 0.5 and 1 hpi, cells were fixed, permeabilized, and stained with anti-NP (red) and DAPI (blue). At least 9 randomly selected fields from each group were analyzed for quantitative co-localization of the nucleus and NP protein. Representative images are shown in (a). Scale bar, 20  μm. Data represent the mean ± SD from one representative experiment of at least three independent experiments, with at least 100 cells per condition quantified. b–e Rab27a-KO and A549-Cas9 cells were pretreated with cycloheximide (CHX, 100 μM) or DMSO (−CHX) for 4 h, followed by infection with HM virus (MOI, 10) and subsequent treatment with CHX (350 μM) or DMSO. The mRNA and vRNA levels of viral genes were quantified by qRT-PCR and normalized to 18S rRNA levels. f A549-Cas9, Rab27a-KO, and Rab27a-KO cells stably expressing Flag-Rab27a-WT, Flag-Rab27a-T23N, or Flag-Rab27a-Q78L were infected with HM virus (MOI, 10). Cells were fixed in glutaraldehyde at 6 hpi, and the budding of influenza viruses was observed using transmission electron microscopy (TEM). For quantitative analysis, 30 cells were randomly selected from each group to determine the number of IAV budding events. Representative images are shown in (f), with scale bars of 5 μm and 1 μm. Data represent the mean ± SD from one representative experiment of at least three independent experiments. Statistical significance was analyzed using a two-tailed Student’s t-test.