Abstract: The objective of this study was to identify the differentially expressed circRNAs in A549 cells upon influenza A virus infection. The findings will provide a basis for future researches on the regulatory roles of circular RNAs (circRNAs) in influenza A virus replication. RNA extracted from IAV-infected A549 cells and PBS-treated control cells were subjected to whole transcriptome sequencing. Bioinformatic analysis was performed to identify the circRNAs expressed in the cells. Comparing with uninfected control samples, differentially expressed circRNAs in cells before and after infection were screened using the EdgeR package with the selection criteria of log₂FC≥1 and P value<0.05. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted on the parental genes of the differentially expressed circRNAs using the ClusterProfiler package. Finally, the differential expression of some circRNAs before and after viral infection was verified by fluorescence quantitative PCR, exonuclease treatment, and Sanger sequencing. The abundance and varity of circRNAs were found to increase upon influenza virus infection. By RNA sequencing, a total of 1 101 and 676 circRNAs were identified in the infected and uninfected groups, respectively. These circRNAs were widely distributed across all chromosomes, with approximately 60% ranging in length from 300 nt to 1 000 nt, and around 20% of circRNAs being longer than 2 000 nt. Furthermore, 75%-82% of circRNAs were derived from exons, while 12%-17% were classified as sense overlapping, and a small fraction (1%-4%) originated from intronic, intergenic, or antisense transcripts. Differential expression analysis identified 54 up-regulated and 18 down-regulated circRNAs in response to viral infection. These differentially expressed circRNAs were mainly distributed on chromosomes 5 and 19. Gene ontology (GO) enrichment analysis revealed that the parent genes of differentially expressed circRNAs were significantly enriched in RNA localization, cytoplasmic membrane, and single-stranded DNA binding. Additionally, KEGG pathway analysis demonstrated that these circRNAs were mainly enriched in pathways related to amino acid degradation and FoxO signaling. Finally, the differential expression of three circRNAs (SNUPN:has_circ_0104558, DCBLD1:hsa_circ_0077717, and EIF4G3:hsa_circ_0000025) was verified by RT-qPCR, RNase R digestion, and Sanger sequencing, which confirmed that they were back spliced from the corresponding source sequences and were resistant to RNase R digestion. The circRNA expression profiles in A549 cells upon influenza A virus infection were obtained using transcriptome sequencing and bioinformatics analysis. The length, chromosome distribution, source classification of these circRNAs, and the potential pathways they probably involved in were analyzed. We identified differentially expressed circRNAs upon viral infection, which lays a foundation for further investigations on the potential roles of circRNAs in influenza virus infection.

Key words: circRNA, influenza A virus, A549 cells, differential expression