Fig. 4. MARCH8 reduces IAV release.

a, b HEK293T cells were transfected with vector, MARCH8 or W114A. Cells were incubated with WSN virus (MOI = 5) at 4 °C for 1 h (a) or then allowed to internalize bound IAV by incubation at 37 °C for another 30 min before adding exogenous NA to remove cell-surface virions. Viral binding or entry was assessed by determining the viral copy number in cell lysates by quantitative real-time PCR. c, d HEK293T cells were co-transfected with pPolI-Luc and expression plasmids containing viral PB1, PB2, PA, or NP of H1N1 along with control (NC) or MARCH8 siRNAs (c), MARCH8 or vector (d). Renilla luciferase was used as an internal control. Luciferase activity was determined at 24 h post transfection. e HEK293T cells transfected with MARCH8 or vector were infected with an MOI of 5 of WSN virus for 10 h and thin sections were analyzed by electron microscopy. The scale bars indicate 200 nm. f–h HEK293T cells were transfected with vector, MARCH8 or W114A. Cells were infected with WSN virus at a MOI of 0.2. At 24 h post infection, HA in the supernatants was determined by ELISA (f), and viral protein expression in cell lysates and the released virus particles were detected by western blotting (g). Quantitation of released NP was shown in (h). Data shown are the mean ± SD (n = three independent experiments). **P < 0.001, n.s., nonsignificant, unpaired two-tailed Student t-test, without any adjustments for multiple comparisons.