IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells.

Susann Kummer,Hans-Georg Kräusslich,Ori Avinoam

doi:10.3390/v11060548

Abstract

Interferon-induced transmembrane proteins (IFITMs) have been shown to strongly affect influenza A virus (IAV) infectivity in tissue culture. Moreover, polymorphisms in IFITM3 have been associated with the severity of the disease in humans. IFITM3 appears to act early in the infection, but its mechanism of action and potential interactions with incoming IAV structures are not yet defined. Here, we visualized endogenous IFITM3 interactions with IAV in the human lung epithelial cell line A549 and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. By applying an iterative approach for the cluster definition and computational cluster analysis, we found that IFITM3 reorganizes into clusters as IAV infection progresses. IFITM3 cluster formation started at 2-3 h post infection and increased over time to finally coat IAV-containing endosomal vesicles. This IAV-induced phenotype was due to the endosomal recruitment of IFITM3 rather than to an overall increase in the IFITM3 abundance. While the IAV-induced IFITM3 clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line A549, the endogenous IFITM3 signal was higher in primary cells. Moreover, we observed IFITM3 signals adjacent to IAV-containing recycling endosomes.

Highlights

Influenza A virus (IAV) is the major cause for a contagious illness of the upper and lower respiratory tract during the seasonal influenza epidemics with peaks in fall and winter for each hemisphere [1,2,3]
The IFITM3 signal increase and clustering correlated with the overall dim NP signals in the extranuclear space and no nuclear NP signals in the respective cells
Given that we used a relatively high MOI of one and that dim NP signals were generally detected in these cells, these observations strongly argue that influenza A virus (IAV) infection was abortive in the cells exhibiting an early accumulation of IFITM3

Summary

Introduction

Influenza A virus (IAV) is the major cause for a contagious illness of the upper and lower respiratory tract during the seasonal influenza epidemics with peaks in fall and winter for each hemisphere [1,2,3]. IAV hijacks cellular import mechanisms to enter the host cell, thereby making use of multiple entry routes. The binding of IAV by the sialylated receptor [8,9] is followed by clathrin-mediated endocytosis [10,11] or micropinocytosis [12]. It is reported that IAV entry is cell-type dependent [13] and that subtypes with a filamentous particle shape preferentially enter host cells via macropinocytosis [14]. Irrespective of the entry mechanism, IAV exploits the non-linear endosomal pathway with its multitude of branches and needs to pass different stages of the endocytic machinery, which is assembled and constantly renewed around the internalized virus particles [17,18,19].

Methods

Results

Conclusion