Identification of influenza virus-encoded vcircRNAs in human A549 cells by RNA-seq

The discovery of noncoding RNAs is reshaping paradigms in molecular biology and virology. Circular RNAs (circRNAs), a class of novel noncoding RNAs, their circular conformation endows these transcripts with critical physiological and pathological roles. Here, we employed circRNAs sequencing technologies, following RNase R treatment on influenza virus-infected human pulmonary epithelial cells to identify and characterize influenza virus-derived circRNAs. Back spicing junctions mapping analysis identified lots of circRNAs from 7 viral gene segment apart from M gene and subsequent experiments confirmed the presence of these IAV-encoded circRNAs in infected cells and mice lung. We found that polymerase genes could encode numerous viral circRNA (vcircRNAs) within infected cells, such as NP and PB1. By vcircRNA overexpression and knockdown with small interfering RNA, we found that these vcircRNAs is required for efficient viral replication. Notably, IAV-derived circRNAs biogenesis was formed with canonical GU/AG splicing mechanism in infected A549 cells. Splicing site mutant viruses displayed a significant decrease in the viral replication in vitro and pathogenicity in mice. Overall, our findings will not only uncover the novel RNA transcripts in IAVs infection cycle, but also provide a new perspective to investigate the crosstalk between host and IAVs from vcircRNAs. To identify and characterize influenza virus-encoded circRNAs，we conducted a circRNA sequencing (circRNA-seq) by applying BGISEQ short-read technology combined with RNase R treatment. PR8/H1N1 virus was used to infect A549 cells at a MOI 2 for 12 and 24 hours, then total RNAs were collected and the ribosomal RNA was removed. The RNA-seq was performed after RNase R treatment.