Analysis of Expression Profiles of Long Noncoding RNAs and mRNAs in A549 Cells Infected with H3N2 Swine Influenza Virus by RNA Sequencing

  • Yina Zhang
  • Tianqi Yu
  • Yingnan Ding
  • Yahui Li
  • Jing Lei
  • Boli Hu
  • Jiyong ZhouEmail author


Long noncoding RNAs (lncRNAs) participate in regulating many biological processes. However, their roles in influenza A virus (IAV) pathogenicity are largely unknown. Here, we analyzed the expression profiles of lncRNAs and mRNAs in H3N2-infected cells and mock-infected cells by high-throughput sequencing. The results showed that 6129 lncRNAs and 50,031 mRNA transcripts in A549 cells displayed differential expression after H3N2 infection compared with mock infection. Among the differentially expressed lncRNAs, 4963 were upregulated, and 1166 were downregulated. Functional annotation and enrichment analysis using gene ontology and Kyoto Encyclopedia of Genes and Genomes databases (KEGG) suggested that target genes of the differentially expressed lncRNAs were enriched in some biological processes, such as cellular metabolism and autophagy. The up- or downregulated lncRNAs were selected and further verified by quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription PCR (RT-PCR). To the best of our knowledge, this is the first report of a comparative expression analysis of lncRNAs in A549 cells infected with H3N2. Our results support the need for further analyses of the functions of differentially expressed lncRNAs during H3N2 infection.


Influenza virus (IAV) Long noncoding RNA (lncRNA) A549 cells High-throughput sequencing 



This study is supported by grants from the National Key technology R&D Program of China (Grant No. 2015BAD12B01), the China Agriculture Research System (Grant No. CARS-40-K13) and the National Science Foundation of China (Grant No. 31502084).

Author Contributions

JZ and YZ designed the experiments and wrote the paper. YZ performed the majority of the experiments. TY and YD analyzed the data. YL prepared the sequencing samples. JL verified sequencing results and BH edited pictures.

Compliance with Ethical Standards

Conflict of interest

All authors declare that they have no conflict of interest.

Animal and Human Rights Statement

This article does not contain any studies with human or animal subjects.

Supplementary material

12250_2019_170_MOESM1_ESM.pdf (210 kb)
Supplementary material 1 (PDF 210 kb)
12250_2019_170_MOESM2_ESM.xlsx (419 kb)
Table S3 Differentially expressed lncRNAs in A549 cells infected with H3N2. (XLSX 418 kb)
12250_2019_170_MOESM3_ESM.xlsx (2.7 mb)
Table S4 Differentially expressed mRNAs in A549 cells infected with H3N2. (XLSX 2799 kb)


  1. Barriocanal M, Carnero E, Segura V, Fortes P (2014) Long non-coding RNA BST2/BISPR is induced by IFN and regulates the expression of the antiviral factor tetherin. Front Immunol 5:655PubMedGoogle Scholar
  2. Carpenter S (2016) Long noncoding RNA: novel links between gene expression and innate immunity. Virus Res 212:137–145CrossRefGoogle Scholar
  3. Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, Byron M, Monks B, Henry-Bezy M, Lawrence JB, O’Neill LA, Moore MJ, Caffrey DR, Fitzgerald KA (2013) A long noncoding RNA mediates both activation and repression of immune response genes. Science 341:789–792CrossRefGoogle Scholar
  4. Costa FF (2010) Non-coding RNAs: meet thy masters. BioEssays 32:599–608CrossRefGoogle Scholar
  5. Dangi T, Jain A (2012) Influenza virus: a brief overview. Proc Natl Acad Sci India Sect B Biol Sci 82:111–121CrossRefGoogle Scholar
  6. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21CrossRefGoogle Scholar
  7. Feng M, Yuan Z, Xia W, Huang X, Wang X, Yan Y, Liao M, Zhou J (2018) Monoclonal antibody against the universal M2 epitope of influenza A virus. Appl Microbiol Biotechnol 102:5645–5656CrossRefGoogle Scholar
  8. Gannage M, Dormann D, Albrecht R, Dengjel J, Torossi T, Ramer PC, Lee M, Strowig T, Arrey F, Conenello G, Pypaert M, Andersen J, Garcia-Sastre A, Munz C (2009) Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes. Cell Host Microbe 6:367–380CrossRefGoogle Scholar
  9. Gualdoni GA, Mayer KA, Kapsch AM, Kreuzberg K, Puck A, Kienzl P, Oberndorfer F, Fruhwirth K, Winkler S, Blaas D, Zlabinger GJ, Stockl J (2018) Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication. Proc Natl Acad Sci USA 115:E7158–E7165CrossRefGoogle Scholar
  10. Horimoto T, Kawaoka Y (2005) Influenza: lessons from past pandemics, warnings from current incidents. Nat Rev Microbiol 3:591–600CrossRefGoogle Scholar
  11. Ilott NE, Heward JA, Roux B, Tsitsiou E, Fenwick PS, Lenzi L, Goodhead I, Hertz-Fowler C, Heger A, Hall N, Donnelly LE, Sims D, Lindsay MA (2014) Long non-coding RNAs and enhancer RNAs regulate the lipopolysaccharide-induced inflammatory response in human monocytes. Nat Commun 5:3979CrossRefGoogle Scholar
  12. Imamura K, Imamachi N, Akizuki G, Kumakura M, Kawaguchi A, Nagata K, Kato A, Kawaguchi Y, Sato H, Yoneda M, Kai C, Yada T, Suzuki Y, Yamada T, Ozawa T, Kaneki K, Inoue T, Kobayashi M, Kodama T, Wada Y, Sekimizu K, Akimitsu N (2014) Long noncoding RNA NEAT1-dependent SFPQ relocation from promoter region to paraspeckle mediates IL8 expression upon immune stimuli. Mol Cell 53:393–406CrossRefGoogle Scholar
  13. Ito T, Couceiro JN, Kelm S, Baum LG, Krauss S, Castrucci MR, Donatelli I, Kida H, Paulson JC, Webster RG, Kawaoka Y (1998) Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 72:7367–7373PubMedPubMedCentralGoogle Scholar
  14. Jiang M, Zhang S, Yang Z, Lin H, Zhu J, Liu L, Wang W, Liu S, Liu W, Ma Y, Zhang L, Cao X (2018) Self-recognition of an inducible host lncRNA by RIG-I feedback restricts innate immune response. Cell 173(906–919):e913Google Scholar
  15. Kong L, Zhang Y, Ye ZQ, Liu XQ, Zhao SQ, Wei L, Gao G (2007) CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res 35:W345–W349CrossRefGoogle Scholar
  16. Lemon SM, Mahmoud AA (2005) The threat of pandemic influenza: are we ready? Biosecur Bioterror 3:70–73CrossRefGoogle Scholar
  17. Li A, Zhang J, Zhou Z (2014) PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme. BMC Bioinform 15:311CrossRefGoogle Scholar
  18. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550CrossRefGoogle Scholar
  19. McArdle J, Schafer XL, Munger J (2011) Inhibition of calmodulin-dependent kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny. J Virol 85:705–714CrossRefGoogle Scholar
  20. Nishitsuji H, Ujino S, Yoshio S, Sugiyama M, Mizokami M, Kanto T, Shimotohno K (2016) Long noncoding RNA #32 contributes to antiviral responses by controlling interferon-stimulated gene expression. Proc Natl Acad Sci USA 113:10388–10393CrossRefGoogle Scholar
  21. Ouyang J, Zhu X, Chen Y, Wei H, Chen Q, Chi X, Qi B, Zhang L, Zhao Y, Gao GF, Wang G, Chen JL (2014) NRAV, a long noncoding RNA, modulates antiviral responses through suppression of interferon-stimulated gene transcription. Cell Host Microbe 16:616–626CrossRefGoogle Scholar
  22. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295CrossRefGoogle Scholar
  23. Qiu L, Wang T, Tang Q, Li G, Wu P, Chen K (2018) Long non-coding RNAs: regulators of viral infection and the interferon antiviral response. Front Microbiol 9:1621CrossRefGoogle Scholar
  24. Simonsen L, Clarke MJ, Williamson GD, Stroup DF, Arden NH, Schonberger LB (1997) The impact of influenza epidemics on mortality: introducing a severity index. Am J Public Health 87:1944–1950CrossRefGoogle Scholar
  25. Simonsen L, Clarke MJ, Schonberger LB, Arden NH, Cox NJ, Fukuda K (1998) Pandemic versus epidemic influenza mortality: a pattern of changing age distribution. J Infect Dis 178:53–60CrossRefGoogle Scholar
  26. Sun L, Luo H, Bu D, Zhao G, Yu K, Zhang C, Liu Y, Chen R, Zhao Y (2013) Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res 41:e166CrossRefGoogle Scholar
  27. Szczesniak MW, Makalowska I (2016) lncRNA-RNA interactions across the human transcriptome. PLoS ONE 11:e0150353CrossRefGoogle Scholar
  28. Tanida I, Ueno T, Kominami E (2008) LC3 and autophagy. Methods Mol Biol 445:77–88CrossRefGoogle Scholar
  29. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, Fukuda K (2003) Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA 289:179–186CrossRefGoogle Scholar
  30. Tong L, Qiu Y, Wang H, Qu Y, Zhao Y, Lin L, Wang Y, Xu W, Zhao W, He H, Zhao G, Zhang MH, Yang D, Ge X, Zhong Z (2019) Expression profile and function analysis of long non-coding RNAs in the infection of coxsackievirus B3. Virol Sin. CrossRefGoogle Scholar
  31. Vastag L, Koyuncu E, Grady SL, Shenk TE, Rabinowitz JD (2011) Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism. PLoS Pathog 7:e1002124CrossRefGoogle Scholar
  32. Vijayan M, Hahm B (2014) Influenza viral manipulation of sphingolipid metabolism and signaling to modulate host defense system. Scientifica (Cairo) 2014:793815Google Scholar
  33. Wang L, Park HJ, Dasari S, Wang S, Kocher JP, Li W (2013) CPAT: coding-potential assessment tool using an alignment-free logistic regression model. Nucleic Acids Res 41:e74CrossRefGoogle Scholar
  34. Wang P, Xu J, Wang Y, Cao X (2017) An interferon-independent lncRNA promotes viral replication by modulating cellular metabolism. Science 358:1051–1055CrossRefGoogle Scholar
  35. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56:152–179PubMedCentralGoogle Scholar
  36. Winterling C, Koch M, Koeppel M, Garcia-Alcalde F, Karlas A, Meyer TF (2014) Evidence for a crucial role of a host non-coding RNA in influenza A virus replication. RNA Biol 11:66–75CrossRefGoogle Scholar
  37. Wu X, Wang H, Bai L, Yu Y, Sun Z, Yan Y, Zhou J (2013) Mitochondrial proteomic analysis of human host cells infected with H3N2 swine influenza virus. J Proteomics 91:136–150CrossRefGoogle Scholar
  38. Xue J, Chambers BS, Hensley SE, Lopez CB (2016) Propagation and characterization of influenza virus stocks that lack high levels of defective viral genomes and hemagglutinin mutations. Front Microbiol 7:326PubMedPubMedCentralGoogle Scholar
  39. Zhirnov OP, Klenk HD (2013) Influenza A virus proteins NS1 and hemagglutinin along with M2 are involved in stimulation of autophagy in infected cells. J Virol 87:13107–13114CrossRefGoogle Scholar
  40. Zhou Z, Jiang XJ, Liu D, Fan Z, Hu XD, Yan JG, Wang M, Gao GF (2009) Autophagy is involved in influenza A virus replication. Autophagy 5:321–328CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS 2019

Authors and Affiliations

  1. 1.MOA Key Laboratory of Animal Virology and Department of Veterinary MedicineZhejiang UniversityHangzhouChina
  2. 2.MOE International Joint Collaborative Research Laboratory for Animal Health and Food Safety, Institute of Immunology and College of Veterinary MedicineNanjing Agricultural UniversityNanjingChina

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