Outbreaks of influenza, caused by the influenza A virus (IAV), occur almost every year in various regions worldwide, seriously endangering human health. Studies have shown that host non-coding RNA is an important regulator of host–virus interactions in the process of IAV infection. In this paper, we comprehensively analyzed the research progress on host non-coding RNAs with regard to the regulation of IAV replication. According to the regulation mode of host non-coding RNAs, the signal pathways involved, and the specific target genes, we found that a large number of host non-coding RNAs directly targeted the PB1 and PB2 proteins of IAV. Nonstructural protein 1 and other key genes regulate the replication of IAV and indirectly participate in the regulation of the retinoic acid-induced gene I-like receptor signaling pathway, toll-like receptor signaling pathway, Janus kinase signal transducer and activator of transcription signaling pathway, and other major intracellular viral response signaling pathways to regulate the replication of IAV. Based on the above findings, we mapped the regulatory network of host non-coding RNAs in the innate immune response to the influenza virus. These findings will provide a more comprehensive understanding of the function and mechanism of host non-coding RNAs in the cellular anti-virus response as well as clues to the mechanism of cell–virus interactions and the discovery of antiviral drug targets.

Increasingly, attention has focused on improving vaccine production in cells using gene editing technology to specifically modify key virus regulation-related genes to promote virus replication. In this study, we used DIA proteomics analysis technology to compare protein expression differences between two groups of MDCK cells: uninfected and influenza A virus (IAV) H1N1-infected cells 16 h post infection (MOI = 0.01). Initially, 266 differentially expressed proteins were detected after infection, 157 of which were upregulated and 109 were downregulated. We screened these proteins to 23 genes related to antiviral innate immunity regulation based on functional annotation database analysis and verified the mRNA expression of these genes using qPCR. Combining our results with published literature, we focused on the proteins RSAD2, KCNN4, IDO1, and ISG20; we verified their expression using western blot, which was consistent with our proteomics results. Finally, we knocked down RSAD2 using lentiviral shRNA expression vectors and found that RSAD2 inhibition significantly increased IAV NP gene expression, effectively promoting influenza virus replication with no significant effect on cell proliferation. These results indicate that RSAD2 is potentially an effective target for establishing high-yield vaccine MDCK cell lines and will help to fully understand the interaction mechanism between host cells and influenza viruses.

The post-translational modification and stability regulation of RIG-I play critical roles in promoting IFN-I production and maintaining immune homeostasis. In this study, we found that ubiquitin-specific peptidase 1 (USP1) promotes RIG-I protein stability through deubiquitination, which in turn enhances antiviral immunity through the production of inflammatory cytokines, and inhibits the replication of influenza virus in MDCK cells. In contrast, USP1 knockdown inhibited the deubiquitination of RIG-I, decreased the RIG-I protein level, and significantly increased the influenza virus titer. Meanwhile, inhibition of USP1 expression did not have a significant effect on the proliferation of MDCK cells, suggesting that USP1 could be used as a target gene to establish a vaccine-producing MDCK cell line. The above results provide a more comprehensive understanding of the function of USP1 and the antiviral response mechanism, and provide a theoretical and methodological basis for the screening of target genes for the artificial establishment of high-yield MDCK cell lines for vaccine production.

The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to the tumorigenicity of MDCK cells. In this study, EGFR-knockdown and EGFR-overexpression cell lines were established. EGFR’s influence on cell growth, migration, clonogenic ability, and flu virus susceptibility was evaluated in vitro, and its role in cell tumorigenicity was examined in nude mice. GST pull-down coupled with mass spectrometry (MS) and bioinformatics analysis identified EGFR-interacting proteins. The expression levels of these proteins, as well as those of PI3K–AKT- and MAPK–ERK-signaling-pathway-related molecules, were confirmed at both gene and protein levels. The result indicates that EGFR overexpression can enhance cell proliferation, migration, and clonal formation; EGFR knockdown could effectively curtail tumorigenesis and amplify the titers of influenza viruses in MDCK cells. An analysis of the underlying mechanism identified a total of 21 interacting proteins implicated in tumor formation, and among these, AKT1, CDK4, GNB2, and MAPK8 were confirmed at both gene and protein levels. EGFR can activate key factors of the PI3K–AKT signaling pathway, AKT and PI3K, and promote their phosphorylation levels. Consequently, we concluded that EGFR interacts with GNB2, facilitating transmembrane signal transduction, activating the PI3K–AKT signaling cascade, controlling cell cycle alterations, stimulating cell proliferation, and promoting tumorigenesis.