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Bovine turbinate cells as an in vitro model for the study of influenza D virus

ABSTRACT: Although it causes only mild respiratory disease, influenza D virus (IDV) is implicated in bovine respiratory disease complex (BRDC), which is a major cause of economic concern for cattle producers globally. The virus replicates in the upper and lower respiratory tract with virus titre highest in nasal turbinates. We therefore hypothesised that the bovine turbinate (BT) cell line could provide a useful in vitro model to study viral–host interactions. Host gene expression was determined using RNA-Seq. Significantly differentially expressed genes with respect to mock-infected cells were identified using DESeq2 and KEGG pathway analysis used to determine enriched pathways. Psuedotyped viruses were used to show the involvement of intrinsic innate immune responses including IFN-inducible transmembrane (IFITM) proteins in restricting the entry of IAV, ICV and IDV. Localization of bovine IFITM3 was determined using immunofluorescence assay. Pull-down and immunoprecipitation assays were used to detect binding of IDV NS1 to p85β subunit of the PI3K pathway. RNA-Seq demonstrated the involvement of genes related to type I or type III interferon responses in IDV infection of BT cells. Intrinsic innate immune responses including IFN-inducible transmembrane (IFITM) proteins restricted the entry of IAV, ICV and IDV pseudotyped viruses. Determination of the subcellular localization of bovine IFITM3 showed that it has an intracellular cytoplasmic distribution. KEGG pathway analysis of the transcriptomic data showed activation of the PI3K/Akt pathway. This led to the hypothesis that IDV activates the pathway in a similar way to influenza A virus (IAV), by non-structural NS1 protein binding the PI3K p85β subunit. However, pull-down and immunoprecipitation assays did not detect binding of IDV NS1 to p85β; this may be due to amino acid differences in the tyrosine-phosphorylated YXXXM motif that is important in the binding of the IAV NS1 to the p85β subunit. This suggests a different mechanism in preventing apoptosis after IDV infection. In conclusion, this study has shown the efficient replication of IDV in bovine turbinate cells. The study has also shown the involvement of type I interferon responses in immune response to IDV infection of cells of the upper respiratory tract of the cow. There is the involvement of different signalling pathways including the FoXO and PI3K/Akt pathway in IDV infection. Bovine IFITM3 is localised intracellularly and is involved in the restriction of IDV entry.

ORGANISM(S): Bos taurus

PROVIDER: GSE252612 | GEO | 2025/01/05

REPOSITORIES: GEO

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Project description:Ifitm3 (interferon-inducible transmembrane protein 3) was previously identified as an endosomal antiviral effector protein that blocks viral infection1-4. In analyses of gene expression data from patients with B-cell leukemia and lymphoma, we identified Ifitm3 as one of the strongest predictors of poor clinical outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, engagement of the B-cell receptor (BCR) and PI3K-activation strongly induced expression of Ifitm3 and phosphorylation of its N-terminus at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases induced phosphorylation at Y20, resulting in constitutive plasma membrane localization of Ifitm3. Ifitm3¯ / ¯ naïve B-cells developed at normal numbers, however, B-cell activation upon antigen encounter, germinal center formation and production of antigen-specific antibodies were compromised. Likewise, oncogenes that typically induce development of leukemia and lymphoma failed to transform Ifitm3¯ / ¯ B-cells. Conversely, a phosphomimetic mutation of Y20 induced oncogenic PI3K-signaling and initiated transformation of premalignant B-cells into overt leukemia. Functional experiments revealed a previously unrecognized function of Ifitm3 as PIP3-scaffold and central amplifier of PI3K signaling downstream of the BCR and adhesion receptors. PI3K signal-amplification depends on binding of Ifitm3 to PIP3 via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3¯ / ¯ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, which impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon Bcell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling downstream of the BCR and adhesion receptors is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3- dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

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Bovine turbinate cells as an in vitro model for the study of influenza D virus

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