EPS8 facilitates uncoating of influenza A virus

Gloria P. Larson; Vy Tran; Shuiqìng Yú; Yíngyún Caì; Christina A. Higgins; Danielle M. Smith; Steven F. Baker; Sheli R. Radoshitzky; Jens H. Kuhn; Andrew Mehle

We probed each successive step that occurs early in the infectious cycle, beginning with viral attachment. Wild-type or edited cells were incubated with bioluminescent virions (PASN) that package nanoluciferase into the viral particle (Tran et al., 2015) . Cells were incubated at 4ËšC to enable binding but prevent internalization of virions, and luciferase activity was assayed from the bound virions. There was no statistical difference in the amount of virus bound to wild type and both EPS8-edited cell lines, indicating EPS8 is not necessary for FLUAV attachment to cells ( Figure 3C ). To ascertain if EPS8 affects HA-mediated entry or the fusion process, we infected cells with FLUAV encoding a different entry protein, FVG-R, a recombinant virus expressing vesicular stomatitis Indiana virus glycoprotein (VSIV-G) instead of HA (Hao et al., 2008) . Viral gene expression decreased in EPS8-edited cells infected with FVG-R compared to wild type cells ( Figure 3D ). This observed decrease in viral gene expression was similar to the decrease demonstrated during infection with bona fide FLUAV ( Figure 2D and 2E) and suggests EPS8 does not specifically target HAmediated entry.