EPS8 Facilitates Uncoating of Influenza A Virus

Abstract

All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We use gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identifies the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirm its importance in enhancing FLUAV infection and titers. Loss of EPS8 does not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associates with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes is significantly delayed in the absence of EPS8. Our study identifies EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.

Keywords: NCI-60; entry; epidermal growth factor receptor pathway substrate 8 (EPS8); gene correlation analysis; influenza virus; uncoating.

Figure 1.. Gene Correlation Analysis Identifies Putative Enhancers and Suppressors of FLUAV Replication.

(A) Experimental workflow for NCI-60 screen. NCI-60 cell lines were inoculated with FLUAV encoding GFP, infections were visualized by fluorescence microscopy and quantified by flow cytometry at 24 hpi, and data were normalized to control MDCK cells inoculated in parallel. (B) Infectivity at an MOI of 0.2 was determined relative to MDCK cells (mean of n = 3 ± SD). Images of highly resistant (MCF7) and hypersensitive (T-47D) infected cell lines are shown compared with the control MDCK cells. Data are representative of two biological replicates. (C) Pairwise comparison of replicate NCI-60 screens performed at an MOI of 0.2 or 2 (mean of n = 3 ± SD; r_s, Spearman’s correlation coefficient). (D) COMPARE analysis of MOI 0.2 infectivity data identified top hits for putative pro-viral and anti-viral factors. See also Figure S1.

Figure 2.. EPS8 Enhances FLUAV Gene Expression and Titers.

(A) 293T cells transiently overexpressing EPS8 were infected with WSN PASTN, and viral gene expression was assayed (mean of n = 4 ± SD). EPS8 expression was confirmed by immunoblot. (B) A549 cells stably overexpressing EPS8 were infected with WSN (MOI 0.01), and viral titer was assayed at 24 hpi (mean of n = 3 ± SD). (C) EPS8-edited A549 cells were infected with WSN PASTN, virus was harvested at the indicated times, and titers were assessed using luciferase activity (mean of n = 3 ± SD). (D and E) EPS8-edited and complemented A549 (D) or 293 (E) cells were infected with WSN PASTN to assay viral gene expression (mean of n = 4 ± SD). EPS8 expression was confirmed by immunoblot. (F) Viral gene expression was assayed in EPS8-edited cells infected with S009 SRK PASTN, CA04 PASTN, or B/Brisbane PASTN (mean of n = 4 ± SD). *p < 0.05 and **p < 0.01; ns, not significant. (A) and (B) were analyzed using Student’s two-tailed t test, unequal variance. Multiple comparisons were made in (C)–(F) using a one-way ANOVA with post hoc Tukey honestly significant difference (HSD) test compared with WT A549 cells. All data are representative of three biological replicates. See also Figures S2–S4.

Figure 3.. EPS8 Functions Post-fusion but before Viral Gene Expression during FLUAV Infection.

(A) Early stages in the FLUAV replication cycle were systematically probed with the indicated reagents or assays detailed in the text. PASN, bioluminescent virions; FVG-R, recombinant FLUAV expressing VSIV-G; IFA, immunofluorescence assay; PAA, polymerase activity assay. (B) Polymerase activity assays were performed in 293T cells expressing RNP components with or without exogenous EPS8. Firefly luciferase (FF) values were normalized to Renilla luciferase (RLuc) values within each sample. EPS8 expression was confirmed by immunoblot. (C) Virion attachment was assayed in EPS8-edited cells incubated with bioluminescent PASN. (D) WT or EPS8-edited cells were inoculated with FVG-R and viral gene expression was measured 8 hpi. (E) Acid bypass assays were performed on virions attached to WT or EPS8-edited cells. Cells were transiently treated with buffers at physiological pH 7.4 to initiate canonical viral entry or acidic pH 5.0 to cause fusion at the cell surface. Viral gene expression was measured 8 h after treatment. For all, data are mean of n = 3 ± SD. **p < 0.01; ns, not significant. (B) was analyzed using Student’s two-tailed t test, unequal variance. Multiple comparisons were made in (C)–(E) using a one-way ANOVA with post hoc Tukey HSD test compared with WT A549 cells. All data are representative of three biological replicates. See also Figure S5.

Figure 4.. EPS8 Is Crucial for Viral Uncoating.

(A) A549 cells synchronously infected with WSN were stained for M1 (red) and the nucleus (blue). Representative images show punctate M1 consistent with intact viral cores and diffuse M1 staining that occurs following viral uncoating. (B) Quantification of diffuse staining in M1-positive cells (mean of n = 2 ± SD). (C) EPS8-edited A549 cells complemented with EPS8 were infected and lysates subjected to immunoprecipitation. Co-precipitating NP and total NP and EPS8 expression were confirmed by immunoblot. (D) WT A549 cells infected with WSN were stained for viral RNPs (green). Representative images show cytoplasmic RNP staining or nuclear RNP staining determined by colocalization with the nucleus (blue). (E) Quantification of the number of cells with nuclear RNP staining at each time point (mean of n = 3 ± SD). *p < 0.05, one-way ANOVA with post hoc Tukey HSD test compared with WT A549 cells. Scale bar, 20 μm. All data are representative of three biological replicates. See also Figure S6.