CD81 is involved in both early and late stages of influenza virus infection.

<p>A) A549 cells were treated with non-targeting control siRNA, or CD81 siRNA1 for 48 hours and immunostained with anti-CD81 antibody. Images are maximum projections of confocal z-stacks. B) A549 cells were treated with control or CD81 siRNA for 48 hours. Cells were harvested for western blotting with indicated antibodies. Tubulin was used as a loading control. C) Influenza virus infection is impaired by CD81 depletion. A549 cells were treated with siRNAs or mock treated for 48 hours and subsequently infected with X-31, WSN, or Udorn at a MOI of <0.1 for 36 hours. The viral titer in the supernatant was determined by plaque assays. The shaded bar indicates the infectivity measured in cells mock treated with a transfection solution that contains no siRNA; the hollow bars indicated the infectivity measured in cells treated with control, non-targeting siRNA; the black solid bars indicate the infectivity measured in cells treated with CD81 siRNA. D) The number of infected cells expressing viral NP is reduced by about 50% upon CD81- knockdown. siRNA-treated A549 cells were infected with WSN, X-31, or Udorn viruses with a MOI of <0.1 for 8 hours without acid bypass and the fraction of cells expressing NP was measured through flow cytometry. E) Viral NP expression is unaffected upon CD81 knockdown when influenza infection is induced by the acid-bypass treatment to eliminate the entry defect. siRNA-treated A549 cells were allowed to bind with WSN or X-31 virus on ice for 1 hour, treated with warm low pH PBS buffer (pH 4.5) for 2 minutes. After 8 hours, cells were collected and stained against NP for flow cytometry analysis. F) Virus titer in the supernatant is reduced by ∼50% or more in CD81-knockdown cells infected by influenza viruses through the acid-bypass treatment. Briefly virus was allowed to bind with control or CD81-knockdown cells on ice for 1 hour. Unbound virus particles were washed out and low pH buffer was added in for 2 minutes to trigger virus fusion at the plasma membrane. At 17 hours post infection, supernatant was collected and the viral titer was assayed by plaque assay. For <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003701#ppat-1003701-g001" target="_blank">figure 1C–1F</a>, the error bars are standard deviation derived from three independent experiments. A two-tailed student <i>t-test</i> was performed for all of the numerical data, and the p value of the data is shown. A p value smaller than 0.05 indicates there is a statistically significant difference.