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

Benfield CT, Smith SE, Wright E, Wash RS, Ferrara F, Temperton NJ, Kellam P - J. Gen. Virol. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, UK cbenfield@rvc.ac.uk.

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Microbat and pig IFITM3 are poly(I : C) responsive. Pig NPTr (a) and microbat (b) cells were either mock treated or stimulated by addition of poly(I : C) to the cell medium (50 µg ml−1) or transfection of poly(I : C) (33 µg ml−1) using Lipofectamine 2000 (microbat cells only). After 7 h, qRT-PCR was used to quantify IFITM3 and the reference gene, GAPDH. The fold change in IFITM3 mRNA is expressed relative to mock-treated cells. Results are shown as means±sd for biological triplicates assayed in duplicate.
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f7: Microbat and pig IFITM3 are poly(I : C) responsive. Pig NPTr (a) and microbat (b) cells were either mock treated or stimulated by addition of poly(I : C) to the cell medium (50 µg ml−1) or transfection of poly(I : C) (33 µg ml−1) using Lipofectamine 2000 (microbat cells only). After 7 h, qRT-PCR was used to quantify IFITM3 and the reference gene, GAPDH. The fold change in IFITM3 mRNA is expressed relative to mock-treated cells. Results are shown as means±sd for biological triplicates assayed in duplicate.

Mentions: Finally, we addressed the contribution of IFITM3 to antiviral responses in pig and microbat cells. Endogenous IFITM3 expression was measured using TaqMan quantitative reverse-transcription PCR (qRT-PCR) designed to specifically detect IFITM3 mRNA and not other IFITM paralogues identified in these cells by RACE. Baseline levels of IFITM3 mRNA were readily detected in both the porcine NPTr cells [threshold cycle (Ct) value of IFITM3 = 22.7; Ct value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) = 21.33] and in microbat lung fibroblasts (Ct value of IFITM3  = 25.8; Ct value of GAPDH = 22.8). IFITM3 induction was assessed in response to the dsRNA analogue poly(I : C), a molecular pattern associated with viral infection, which is recognized by Toll-like receptor 3 and induces type I IFN in porcine (Provost et al., 2012) and bat (Biesold et al., 2011; Omatsu et al., 2008; Zhou et al., 2011) cells. Poly(I : C) addition led to a 3.5-fold increase in pig IFITM3 and a twofold increase in microbat IFITM3 mRNA levels (Fig. 7). The microbat cells were also stimulated via poly(I : C) transfection; as in pteropid bat lung cells (but not primary cells from other tissues) this enhanced IFN-β induction relative to extracellular delivery of poly(I : C) (Zhou et al., 2011). However, transfection of poly(I : C) into microbat cells resulted in a similar degree of IFITM3 induction (2.3-fold) (Fig. 7).


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

Benfield CT, Smith SE, Wright E, Wash RS, Ferrara F, Temperton NJ, Kellam P - J. Gen. Virol. (2015)

Microbat and pig IFITM3 are poly(I : C) responsive. Pig NPTr (a) and microbat (b) cells were either mock treated or stimulated by addition of poly(I : C) to the cell medium (50 µg ml−1) or transfection of poly(I : C) (33 µg ml−1) using Lipofectamine 2000 (microbat cells only). After 7 h, qRT-PCR was used to quantify IFITM3 and the reference gene, GAPDH. The fold change in IFITM3 mRNA is expressed relative to mock-treated cells. Results are shown as means±sd for biological triplicates assayed in duplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631062&req=5

f7: Microbat and pig IFITM3 are poly(I : C) responsive. Pig NPTr (a) and microbat (b) cells were either mock treated or stimulated by addition of poly(I : C) to the cell medium (50 µg ml−1) or transfection of poly(I : C) (33 µg ml−1) using Lipofectamine 2000 (microbat cells only). After 7 h, qRT-PCR was used to quantify IFITM3 and the reference gene, GAPDH. The fold change in IFITM3 mRNA is expressed relative to mock-treated cells. Results are shown as means±sd for biological triplicates assayed in duplicate.
Mentions: Finally, we addressed the contribution of IFITM3 to antiviral responses in pig and microbat cells. Endogenous IFITM3 expression was measured using TaqMan quantitative reverse-transcription PCR (qRT-PCR) designed to specifically detect IFITM3 mRNA and not other IFITM paralogues identified in these cells by RACE. Baseline levels of IFITM3 mRNA were readily detected in both the porcine NPTr cells [threshold cycle (Ct) value of IFITM3 = 22.7; Ct value of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) = 21.33] and in microbat lung fibroblasts (Ct value of IFITM3  = 25.8; Ct value of GAPDH = 22.8). IFITM3 induction was assessed in response to the dsRNA analogue poly(I : C), a molecular pattern associated with viral infection, which is recognized by Toll-like receptor 3 and induces type I IFN in porcine (Provost et al., 2012) and bat (Biesold et al., 2011; Omatsu et al., 2008; Zhou et al., 2011) cells. Poly(I : C) addition led to a 3.5-fold increase in pig IFITM3 and a twofold increase in microbat IFITM3 mRNA levels (Fig. 7). The microbat cells were also stimulated via poly(I : C) transfection; as in pteropid bat lung cells (but not primary cells from other tissues) this enhanced IFN-β induction relative to extracellular delivery of poly(I : C) (Zhou et al., 2011). However, transfection of poly(I : C) into microbat cells resulted in a similar degree of IFITM3 induction (2.3-fold) (Fig. 7).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, UK cbenfield@rvc.ac.uk.

Show MeSH
Related in: MedlinePlus