Overexpression of Chicken IRF7 Increased Viral Replication and Programmed Cell Death to the Avian Influenza Virus Infection Through TGF-Beta/FoxO Signaling Axis in DF-1

Abstract

During mammalian viral infections, interferon regulatory factor 7 (IRF7) partners with IRF3 to regulate the type I interferon response. In chickens, however, it is still unclear how IRF7 functions in the host innate immune response, especially given that IRF3 is absent. To further elucidate the functional role of chicken IRF7 during avian influenza virus (AIV) infection, we generated inducible IRF7 overexpression DF-1 cell lines and performed in vitro infection using low pathogenic AIVs (LPAIVs). Overexpression of IRF7 resulted in higher viral replication of H6N2 and H10N7 LPAIVs compared to empty vector control cells regardless of IRF7 expression level. In addition, a high rate of induced cell death was observed due to elevated level of IRF7 upon viral infection. RNA-seq and subsequent transcriptome analysis of IRF7 overexpression and control cells discovered candidate genes possibly controlled by chicken IRF7. Functional annotation revealed potential pathways modulated by IRF7 such as TGF-beta signaling pathway, FoxO signaling pathway and cell structural integrity related pathways. Next, we analyzed the host response alteration due to the IRF7 overexpression and additionally discovered the possible connection of chicken IRF7 and JAK-STAT signaling pathway. These findings suggest that chicken IRF7 could modulate a wide range of cellular processes in the host innate immune response thus meticulous control of IRF7 expression is crucial to the host in response to AIV infection.

Knockout of IRF7 Highlights its Modulator Function of Host Response Against Avian Influenza Virus and the Involvement of MAPK and TOR Signaling Pathways in Chicken

“…In our previous studies, we consistently observed a significant upregulation of IFNB expression as a result of IRF7 overexpression in the DF-1 cell lines upon dsRNA induction or H6N2 infection [14,35]. On the other hand, IFNA mRNA levels were not significantly affected by the level of IRF7 expression [14,35], and a similar pattern was observed in this study ( Figure 1E-G). Although DF-1 cells are capable of mounting a type I IFN response, the level of response is possibly lower than that of primary CEFs due to a higher suppressor of cytokine signaling 1 (SOCS1) expression in DF-1 [42].…”

“…The type I IFN induction models from in vitro studies using mouse embryonic fibroblast or primary tracheal epithelial cells of Irf7 -/mice showed an abolished production of type I IFNs upon viral infection [10,41]. In our previous studies, we consistently observed a significant upregulation of IFNB expression as a result of IRF7 overexpression in the DF-1 cell lines upon dsRNA induction or H6N2 infection [14,35]. On the other hand, IFNA mRNA levels were not significantly affected by the level of IRF7 expression [14,35], and a similar pattern was observed in this study ( Figure 1E-G).…”

“…Accordingly, we observed an expression pattern shift of PI3K and upregulation of AKT3 in the IRF7 -/cells compared to in the IRF7 wt ( Figure S3). Our previous study provided another line of evidence supporting the mTOR-mediated IRF7 modulation of the host response, in which GO terms such as arginine and proline metabolism, pyrimidine metabolism and the regulation of the actin cytoskeleton were significantly enriched from the DEGs of IRF7 overexpression vs. wildtype [35].…”

“…A schematic model of early host response associated with chicken IRF7 is proposed in Figure 6, based on the current study and our previous in vitro studies [14,35]. The entry of AIV is recognized by pattern recognition receptors (PRRs) sensing viral moieties.…”

“…Our previous work has identified candidate genes that were regulated by IRF7 in the context of H6N2 infection by applying an inducible overexpression-based gain-of-function approach [35]. We infected IRF7 overexpression cells (CuO-IRF7: 2-fold IRF7 overexpression) and their respective controls with H6N2 (1 MOI) and performed RNA-seq at 0 h (mock) and 6 hpi.…”