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

Our line of experimentation indicated EPS8 functions at a step following release of the viral core into the cytoplasm but before viral gene expression. Therefore, we considered whether EPS8 facilitates viral uncoating. This process can be quantified by visualizing the redistribution of punctate matrix protein (M1) staining of intact particles to diffuse staining of M1 released throughout the cytosol ( Figure 4A and S5A) (Banerjee et al., 2013) . Wild type and EPS8edited cells were synchronously infected, and M1 localization was quantified at various times postinoculation. As expected, most M1 staining was punctate in wild type cells early in infection and then became diffuse at 1.5 hpi ( Figure 4B ). By contrast, uncoating was greatly delayed in both cell lines where EPS8 was edited. Diffuse M1 staining was detected in only 10-15% of EPS8-edited cells at 1.5 hpi compared to successful uncoating in almost all wild type cells at the same time point. Following release from the endosome, viral cores are trafficked to the aggresome to complete uncoating (Banerjee et al., 2014) . Coprecipitations were used to probe how EPS8 might function during this period. Synchronized infections were initiated on EPS8-edited cells stably complemented with wild type EPS8. NP specifically co-precipitated with EPS8, suggesting EPS8 physically interacts with incoming viral cores ( Figure 4C ). These data implicate EPS8 as an important host factor during viral uncoating. Uncoating releases vRNPs into the cytosol where they are subsequently imported into the nucleus prior to viral gene expression. The defects in uncoating we detected in EPS8-edited cells predict that these cells should also exhibit delayed nuclear import. To test this possibility, we again used synchronized infections and immunofluorescence to examine the subcellular localization and kinetics of vRNP nuclear import over time. Staining for NP, the major protein component of vRNPs, revealed characteristic cytoplasmic localization of incoming vRNPs early during infection followed by distinct nuclear localization ( Figure 4D ). Discrete cytoplasmic and nuclear localizations of vRNPs were also detected in EPS8-edited cells ( Figure S5B ). Nuclear-localized vRNPs were detected in wild type cells as early as 1.5 hpi and the number of cells with nuclear vRNP staining increased over time, consistent with the timing of viral uncoating reported above ( Figure 4E ). Cells lacking wild type levels of EPS8, however, exhibited significantly delayed kinetics of nuclear import. Compared to wild type cells, import rates in EPS8edited cells were delayed by 1 hour. This trend continued until 3.5 hpi when import in edited cells finally matched that of wild type cells ( Figure 4D ). While import was delayed in edited cells, it followed a similar trajectory to wild type cells once initiated, suggesting that vRNP import was not directly altered by changes to EPS8 expression. Thus, defects in uncoating ( Figure 4B ) result in delayed nuclear import ( Figure 4E ) and ultimately a reduction in viral gene expression ( Figure 2D ), reinforcing the conclusion that EPS8 is a key component of the cellular machinery utilized for viral uncoating.