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

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.

Current Progress on Host Antiviral Factor IFITMs

“…A highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 plays a critical role in IFITM3dependent inhibition of IAV, Zika virus, VSV, EBOV, HIV, and SARS-CoV-2 infections (43,71). IFITM3 may accumulate and locate in endosomal vesicles during IAV infection and eventually coat the IAV-containing endosomal vesicles (76). Moreover, IFITM3 can fuse with the incoming virus and enhance the trafficking of the IFITM-virus cargo to the lysosomes for degradation via specific S-palmitoylation (76,77).…”

“…IFITM3 may accumulate and locate in endosomal vesicles during IAV infection and eventually coat the IAV-containing endosomal vesicles (76). Moreover, IFITM3 can fuse with the incoming virus and enhance the trafficking of the IFITM-virus cargo to the lysosomes for degradation via specific S-palmitoylation (76,77).…”

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

“…It was previously reported that IFITM3 forms clusters on virus-containing vesicles ( Kummer et al, 2019 ) and that IFITM3 oligomerization promotes restriction of IAV ( John et al, 2013 ). However, the determinants initially purported to mediate oligomerization (phenylalanines at residues 75 and 78 of the CD225 domain [ John et al, 2013 ]) were later shown to be unnecessary when oligomerization was measured using a FRET-based approach in living cells ( Winkler et al, 2019 ).…”

Investigation of Human IFITM3 Polymorphisms rs34481144A and rs12252C and Risk for Influenza A(H1N1)pdm09 Severity in a Brazilian Cohort

“…The antiviral restriction factor IFITM3 acts as part of innate immune response initial steps to limit influenza infection. It halts the formation of viral membrane fusion pores with host endosomes, by clustering in the IAV containing endosomes, impairing entry into the cytosol and subsequent viral replication (Kummer et al, 2019 ; Suddala et al, 2019 ). In addition, IFITM3 has also been shown to be induced, in DCs, after influenza infection, in mice, and is involved in migration of these cells to lymph nodes and consequent activation of influenza-specific lymphocytes (Infusini et al, 2015 ).…”