Pro-inflammatory Effects of Influenza Type A Virus PB1-F2 Protein-derived Peptide in Lipopolysaccharide-treated Macrophages

  • Kurosh Kalantar Mail Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
  • Zahra Farzaneh Department of Immunology, Medical School, Shiraz University of Medical Sciences, Shiraz, Iran
  • Nasser Gholijani Autoimmune Diseases Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
  • Seyed Younes Hosseini Department of Bacteriology and Virology, Shiraz University of Medical Sciences, Shiraz, Iran
  • Ebrahim Bani Hasan Australian Institute for Musculoskeletal Science (AIMSS), The University of Melbourne and Western Health, St. Albans, VIC, Australia AND Department of Medicine-Western Health, The University of Melbourne, St. Albans, VIC, Australia
Inflammation, Influenza A virus, Macrophage, Polymerase basic–frame-2


Influenza A virus (IAV) has the potential to cause pandemics with considerable health and socio-economic burdens. A viral protein, polymerase basic 1- frame2 (PB1-F2), as a virulence factor, has pro-apoptotic activity and contributes to viral pathogenesis by delaying viral clearance and inducing inflammation. Macrophages are susceptible to IAV infection and produce high levels of inflammatory cytokines and chemokines. In the present study, the pro-inflammatory effects of PB1-F2 derived peptide was evaluated by measuring the expression of key inflammatory mediators in murine macrophage cell line J774.1.
PB1-F2 treated macrophages were examined for nitric oxide (NO) production, inflammatory cytokines, and enzymes expression and pro-inflammatory cytokines secretion using Griess reagent, real-time polymerase chain reaction (PCR) and ELISA, respectively. Our results have shown that PB1-F2 peptide at non-cytotoxic concentrations (0.1–0.8 µmol/mL) had no effect on NO production.
When applied to Lipopolysaccharide (LPS)-treated macrophages, PB1-F2 peptide at 0.8 μmol/mLincreasedinducible NO synthase (iNOS), cyclooxygenase (COX)-2, and interleukin (IL)-6 genes expression to 2.02, 3.81, and 3.65 folds, respectively. PB1-F2 at concentrations of 0.4 and 0.8 µm/mL increased tumor necrosis factor (TNF)-α transcription by 4.15 and 5.55 fold. At posttranslational level, TNF-α increased from 166.5±13.88 in LPS-treated cells to 773.6±95.27 and 1485±76.31 at concentrations of 0.4 and 0.8 μmol/mL in PB1-F2 peptide, respectively. However, PB1-F2 Peptide did not have any significant effect on IL-6 production.
These findings suggest that PB1-F2 peptide may partly exert its enhancing role in viral pathogenicity through the induction of inflammatory mediators in macrophages. Hence, targeting PB1-F2 peptide would be helpful in the reduction of viral infection complications.


1. Perrone LA, Plowden JK, et al. H5N1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice. PLoS pathogens. 2008;4(8):e1000115.
2. BaskinCR, Bielefeldt-Ohmann H, Tumpey TM, Sabourin PJ, Long JP, García-Sastre A, et al. Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus. Proc Natl Acad Sci U S A. 2009;106(9):3455-60.
3. Conn CA, McClellan J, Maassab H, et al. Cytokines and the acute phase response to influenza virus in mice. Am J Physiol. 1995;268(1):R78-R84.
4. Vacheron F, Rudent A, Perin S, Labarre C, Quero A, Guenounou M. Production of interleukin 1 and tumour necrosis factor activities in bronchoalveolar washings following infection of mice by influenza virus. J Gen Virol. 1990;71(2):477-9.
5. Huo C, Xiao K, Zhang S, Tang Y, Wang M, Qi P, et al. H5N1 influenza A virus replicates productively in pancreatic cells and induces apoptosis and pro-inflammatory cytokine response. Front Cell Infect Microbiol. 2018;8:386.
6. Varga ZT, Palese P. The influenza A virus protein PB1-F2: killing two birds with one stone? Virulence. 2011;2(6):542-6.
7. Chen W, Calvo PA, Malide D, Gibbs J, Schubert U, Bacik I, et al. A novel influenza A virus mitochondrial protein that induces cell death. Nat Med. 2001;7(12):1306-12.
8. Alymova IV, Green AM, van de Velde N, McAuley JL, Boyd KL, Ghoneim H, et al. Immunopathogenic and anti-bacterial effects of the H3N2 influenza A virus PB1-F2 map to amino acid residues 62, 75, 79,and 82. J Virol. 2011:JVI. 05872-11.
9. Kamal RP, Alymova IV, York IA. Evolution and Virulence of Influenza A Virus Protein PB1-F2. Int J Mol Sci. 2017;19(1):96.
10. Krumbholz A, Philipps A, Oehring H, Schwarzer K, Eitner A, Wutzler P, et al. Current knowledge on PB1-F2 of influenza A viruses. Med Microbiol Immunol. 2011;200(2):69-75.
11. Chakrabarti AK, Pasricha G. An insight into the PB1F2 protein and its multifunctional role in enhancingthe pathogenicity of the influenza A viruses. Virology. 2013;440(2):97-104.
12. Zamarin D, Ortigoza MB, Palese P. Influenza A virus PB1-F2 protein contributes to viral pathogenesis in mice. J Virol. 2006;80(16):7976-83.
13 .Zamarin D, García-Sastre A, Xiao X, Wang R, Palese P. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1. PLoS pathogens. 2005;1(1):e4.
14. McAuley JL, Chipuk JE, Boyd KL, Van De Velde N, Green DR, McCullers JA. PB1-F2 proteins from H5N1and 20th century pandemic influenza viruses cause immunopathology. PLoS pathogens. 2010;6(7):e1001014.
15. McAuley JL, Hornung F, Boyd KL, Smith AM, McKeon R, Bennink J, et al. Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and secondary bacterial pneumonia. Cell Host Microbe. 2007;2(4):240-9.
16. Leymarie O, Embury-Hyatt C, Chevalier C, Jouneau L, Moroldo M, Da Costa B, et al. PB1-F2 attenuates virulence of highly pathogenic avian H5N1 influenza virus in chickens. PloS one. 2014;9(6):e100679.
17. Conenello GM, Tisoncik JR, Rosenzweig E, Varga ZT, Palese P, Katze MG. A single N66S mutation in the PB1-F2 protein of influenza A virus increases virulence by inhibiting the early interferon response in vivo. J Virol. 2011;85(2):652-62.
18. Dudek SE, Wixler L, Nordhoff C, Nordmann A, Anhlan D, Wixler V, et al. The influenza virus PB1-F2 protein has interferon antagonistic activity. Biol Chem. 2011;392(12):1135-44.
19. Farzaneh Z, Kalantar K, Iraji A, Amirghofran Z. Inhibition of LPS-induced inflammatory responses by Satureja hortensis extracts in J774.1 macrophages. J Immunoassay Immunochem. 2018;39(3):274-91.
20. Koussounadis A, Langdon S.P, Um I.H, Harrison D.J, Smith V.A. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system. Sci Rep. 2015;5: 10775.
21. Hofmann P, Sprenger H, Kaufmann A, Bender A, Hasse C, Nain M, et al. Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response. J Leukoc Biol. 1997;61(4):408-14.
22. Matikainen S, Pirhonen J, Miettinen M, Lehtonen A, Govenius-Vintola C, Sareneva T, et al. Influenza A and sendai viruses induce differential chemokine gene expression and transcription factor activation in human macrophages. Virology. 2000;276(1):138-47.
23. Huo C, Jin Y, Zou S, Qi P, Xiao J, Tian H, et al. Lethal influenza A virus preferentially activates TLR3 and triggers a severe inflammatory response. Virus Res. 2018;257:102-12.
24. Pinar A, Dowling JK, Bitto NJ, Robertson AA, Latz E, Stewart CR, et al. PB1-F2 Peptide Derived from Avian Influenza A Virus H7N9 Induces Inflammation via Activation of the NLRP3 Inflammasome. J Biol Chem. 2017;292(3):826-36.
25. Le Goffic R, Bouguyon E, Chevalier C, Vidic Jet al. Influenza A virus protein PB1-F2 exacerbates IFN-β expression of human respiratory epithelial cells. J Immunol. 2010:0903952.
26. Le Goffic R, Leymarie O, Chevalier C, Rebours E, Da Costa B, Vidic J, et al. Transcriptomic analysis of host immune and cell death responses associated with the influenza A virus PB1-F2 protein. PLoS pathogens. 2011;7(8):e1002202.
27. Reis AL, McCauley JW. The influenza virus protein PB1-F2 interacts with IKKβ and modulates NF-κBsignalling. PLoS One. 2013;8(5):e63852.
28. Varga ZT, Grant A, Manicassamy B, Palese P. Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential. J Virol. 2012;86(16):8359-66.
29. Conenello GM, Zamarin D, Perrone LA, Tumpey T, Palese P. A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence. PLoS pathogens. 2007;3(10):e141.
30. Akaike T, Maeda H. Nitricoxide and virus infection. Immunology. 2000;101(3):300-8.
31. Perrone LA, Belser JA, Wadford DA, Katz JM, Tumpey TM. Inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice. J Infect Dis. 2013;207(10):1576-84.
32. Asano K, Chee C, Gaston B, Lilly CM, Gerard C, Drazen JM, et al. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc Natl Acad Sci U S A. 1994;91(21):10089-93.
33. Burggraaf S, Bingham J, Payne J, Kimpton WG, Lowenthal JW, Bean AG. Increased inducible nitric oxide synthase expression in organs is associated with a higher severity of H5N1 influenza virus infection. PloS one. 2011;6(1):e14561.
34. Aoki T, Narumiya S. Prostaglandins and chronic inflammation. Trends in pharmacological sciences. 2012;33(6):304-11.
35. Lee SM, Cheung C-Y, Nicholls JM, Hui KP, Leung CY, Uiprasertkul M, et al. Hyperinduction of cyclooxygenase-2-mediated proinflammatory cascade: a mechanism for the pathogenesis of avian influenza H5N1 infection. J Infect Dis. 2008;198(4):525-35.
36. Mizumura K, Hashimoto S, Maruoka S, Gon Y, Kitamura N, Matsumoto K, et al. Role of mitogen‐activated protein kinases in influenza virus induction of prostaglandin E2 from arachidonic acid in bronchial epithelial cells. Clin Exp Allergy. 2003;33(9):1244-51.
37. Dudek SE, Nitzsche K, Ludwig S, Ehrhardt C. Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability. Sci Rep. 2016;6:27275.
38. Kang Y-J, Mbonye UR, DeLong CJ, Wada M, Smith WL. Regulation of intracellular cyclooxygenase levels by gene transcription and protein degradation. Prog Lipid Res. 2007;46(2):108-25.
How to Cite
Kalantar K, Farzaneh Z, Gholijani N, Hosseini SY, Bani Hasan E. Pro-inflammatory Effects of Influenza Type A Virus PB1-F2 Protein-derived Peptide in Lipopolysaccharide-treated Macrophages. Iran J Allergy Asthma Immunol. 19(S1):74-82.
Original Article(s)