Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by influenza virus and lyssaviruses.

Sarah E Smith,Francesca Ferrara,Paul Kellam,Nigel J Temperton,Rachael S Wash,Camilla T O Benfield,Edward Wright

doi:10.1099/vir.0.000058

Abstract

IFN-induced transmembrane protein 3 (IFITM3) is a restriction factor that blocks cytosolic entry of numerous viruses that utilize acidic endosomal entry pathways. In humans and mice, IFITM3 limits influenza-induced morbidity and mortality. Although many IFITM3-sensitive viruses are zoonotic, whether IFITMs function as antiviral restriction factors in mammalian species other than humans and mice is unknown. Here, IFITM3 orthologues in the microbat (Myotis myotis) and pig (Sus scrofa domesticus) were identified using rapid amplification of cDNA ends. Amino acid residues known to be important for IFITM3 function were conserved in the pig and microbat orthologues. Ectopically expressed pig and microbat IFITM3 co-localized with transferrin (early endosomes) and CD63 (late endosomes/multivesicular bodies). Pig and microbat IFITM3 restricted cell entry mediated by multiple influenza haemagglutinin subtypes and lyssavirus glycoproteins. Expression of pig or microbat IFITM3 in A549 cells reduced influenza virus yields and nucleoprotein expression. Conversely, small interfering RNA knockdown of IFITM3 in pig NPTr cells and primary microbat cells enhanced virus replication, demonstrating that these genes are functional in their species of origin at endogenous levels. In summary, we showed that IFITMs function as potent broad-spectrum antiviral effectors in two mammals – pigs and bats – identified as major reservoirs for emerging viruses.

Highlights

Restriction factors are germline-encoded proteins that function in a cell-autonomous manner to suppress virus replication
Sequence analysis of IFN-induced transmembrane protein 3 (IFITM3) orthologues cloned from microbat and pig cells
It was not possible to assign pig and bat IFITM3 orthologues based on conserved synteny due to the lack of a B4GALNT4 orthologue in pigs, gaps in the genome assemblies and the low sequencing coverage of the bat genomes (2.66 for Pteropus vampyrus and 1.76 for Myotis lucifugus at the time of analysis)

Summary

Introduction

Restriction factors are germline-encoded proteins that function in a cell-autonomous manner to suppress virus replication. The IFN-induced transmembrane (IFITM) proteins are a family of small IFN-stimulated proteins that affect diverse cellular processes (reviewed by Siegrist et al, 2011) and were recently identified as antiviral restriction factors that inhibit the cell entry of multiple pathogenic viruses (Brass et al, 2009). The common feature of IFITM-sensitive viruses appears to be their dependence on acidic endosomal entry pathways, either for proteolytic cleavage, or for pH- or protease-dependent activation of viral entry proteins into their fusogenic form. O. Benfield and others recapitulate the IFITM sensitivity of the authentic virus from which the envelope protein derives, and have been widely used to study IFITM biology (Brass et al, 2009; Feeley et al, 2011; Huang et al, 2011). IFITMs localize to membranes of late endosomes and lysosomes and prevent the release of viral particles from these compartments into the cytosol (Feeley et al, 2011)

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