Host miR-140-5p Directly Targets IFIT2 and Enhances Replication of a Zoonotic Reassortant Swine H3N2 Influenza Virus

Reassortment between the 2009 pandemic H1N1 virus (pdm/09) and endemic swine influenza viruses has generated zoonotic viruses, including A/Swine/Guangxi/NS2783/2010 (H3N2; SW2783). How host microRNAs shape the outcome of such transmission remains poorly defined. We combined iTRAQ-based quantitative proteomics of A549 cells infected with SW2783, the classical swine H1N1 virus SW3861, or the pdm/09 strain CA09 with our previous miRNA-mRNA dataset. Among 2398 proteins consistently quantified, an integrative analysis identified 118 candidate proteins (49, downregulated and 69 upregulated), of which eight, including PPIA and IFIT2, were prioritized as potential virus-response factors. Western blotting showed that RPS29, HMGA2, IFIT2, and PPIA were specifically upregulated in SW2783-infected cells. miR-140-5p reduced nPPIA and IFIT2 protein levels, and dual-luciferase assays supported direct regulation of the IFIT2 3′ UTR, whereas PPIA appeared to be regulated indirectly. Overexpression of miR-140-5p increased SW2783 M-gene transcripts, HA titers, and TCID50 values (p < 0.01) but did not affect replication of SW3861 or CA09. Together, these findings suggest that miR-140-5p promotes replication of the zoonotic reassortant swine H3N2 virus SW2783, at least in part through direct suppression of IFIT2, while its effect on PPIA appears to be indirect. These results identify the miR-140-5p/IFIT2 axis as a potential host regulatory pathway associated with replication of reassortant swine influenza viruses and warrant further investigation of its relevance to zoonotic risk.