Virologica Sinica

, Volume 33, Issue 5, pp 440–448 | Cite as

CypA Regulates AIP4-Mediated M1 Ubiquitination of Influenza A Virus

  • Madina Mahesutihan
  • Weinan Zheng
  • Liang Cui
  • Yun Li
  • Pengtao Jiao
  • Wenxian Yang
  • Wei Liu
  • Jing Li
  • Wenhui Fan
  • Limin Yang
  • Wenjun Liu
  • Lei Sun
Research Article


Cyclophilin A (CypA) is a peptidyl-prolyl cis/trans isomerase that interacts with the matrix protein (M1) of influenza A virus (IAV) and restricts virus replication by regulating the ubiquitin–proteasome-mediated degradation of M1. However, the mechanism by which CypA regulates M1 ubiquitination remains unknown. In this study, we reported that E3 ubiquitin ligase AIP4 promoted K48-linked ubiquitination of M1 at K102 and K104, and accelerated ubiquitin–proteasome-mediated degradation of M1. The recombinant IAV with mutant M1 (K102R/K104R) could not be rescued, suggesting that the ubiquitination of M1 at K102/K104 was essential for IAV replication. Furthermore, CypA inhibited AIP4-mediated M1 ubiquitination by impairing the interaction between AIP4 and M1. More importantly, both the mutations of M1 (K102R/K104R) and CypA inhibited the nuclear export of M1, indicating that CypA regulates the cellular localization of M1 via inhibition of AIP4-mediated M1 ubiquitination at K102 and K104, which results in the reduced replication of IAV. Collectively, our findings reveal a novel ubiquitination-based mechanism by which CypA regulates the replication of IAV.


Influenza A virus (IAV) Ubiquitination Cyclophilin A (CypA) AIP4 M1 



This work was supported by grants from the National Natural Science Foundation of China (31630079, 31672531, 31572526, and 31802164), the National Key R&D Program of China (2016YFD0500206, 2015BAD11B02), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB29010000), the National Science and Technology Major Project (2018ZX10101004), and the Emergency Technology Research Issue on Prevention and Control for Human Infection with A (H7N9) Avian Influenza Virus (10600100000015001206). Wenjun Liu is the principal investigator of the Innovative Research Group of National Natural Science Foundation of China (Grant No. 81621091).

Author Contributions

LS and Wenjun Liu supervised the project, designed the study, and analyzed the data; MM and LS were responsible for planning and conducting the experimental work, and wrote the manuscript; WZ, Wei Liu and WF provided the technical support; LC, YL, PJ and WY participated in part of experimental work. JL and LY analyzed the data.

Compliance with Ethical Standards

Conflict of interest

The 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 performed by any of the authors.


  1. Arranz R, Coloma R, Chichon FJ, Conesa JJ, Carrascosa JL, Valpuesta JM, Ortin J, Martin-Benito J (2012) The structure of native influenza virion ribonucleoproteins. Science 338:1634–1637CrossRefGoogle Scholar
  2. Baudin F, Petit I, Weissenhorn W, Ruigrok RW (2001) In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology 281:102–108CrossRefGoogle Scholar
  3. Bui M, Wills EG, Helenius A, Whittaker GR (2000) Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol 74:1781–1786CrossRefGoogle Scholar
  4. Burleigh LM, Calder LJ, Skehel JJ, Steinhauer DA (2005) Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J Virol 79:1262–1270CrossRefGoogle Scholar
  5. Calder LJ, Wasilewski S, Berriman JA, Rosenthal PB (2010) Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci USA 107:10685–10690CrossRefGoogle Scholar
  6. Calistri A, Munegato D, Carli I, Parolin C, Palu G (2014) The ubiquitin-conjugating system: multiple roles in viral replication and infection. Cells 3:386–417CrossRefGoogle Scholar
  7. Cao S, Liu X, Yu M, Li J, Jia X, Bi Y, Sun L, Gao GF, Liu W (2012) A nuclear export signal in the matrix protein of influenza A virus is required for efficient virus replication. J Virol 86:4883–4891CrossRefGoogle Scholar
  8. Chau V, Tobias JW, Bachmair A, Marriott D, Ecker DJ, Gonda DK, Varshavsky A (1989) A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science 243:1576–1583CrossRefGoogle Scholar
  9. Di Pietro A, Kajaste-Rudnitski A, Oteiza A, Nicora L, Towers GJ, Mechti N, Vicenzi E (2013) TRIM22 inhibits influenza A virus infection by targeting the viral nucleoprotein for degradation. J Virol 87:4523–4533CrossRefGoogle Scholar
  10. Finley D (2009) Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu Rev Biochem 78:477–513CrossRefGoogle Scholar
  11. Gao S, Wu J, Liu RY, Li J, Song L, Teng Y, Sheng C, Liu D, Yao C, Chen H, Jiang W, Chen S, Huang W (2015) Interaction of NS2 with AIMP2 facilitates the switch from ubiquitination to SUMOylation of M1 in influenza A virus-infected cells. J Virol 89:300–311CrossRefGoogle Scholar
  12. Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW (1984) Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:544–547CrossRefGoogle Scholar
  13. Khor R, McElroy LJ, Whittaker GR (2003) The ubiquitin-vacuolar protein sorting system is selectively required during entry of influenza virus into host cells. Traffic 4:857–868CrossRefGoogle Scholar
  14. Kirui J, Mondal A, Mehle A (2016) Ubiquitination up-regulates influenza virus polymerase function. J Virol. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Klumpp K, Ruigrok RW, Baudin F (1997) Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. EMBO J 16:1248–1257CrossRefGoogle Scholar
  16. Koestler TP, Rieman D, Muirhead K, Greig RG, Poste G (1984) Identification and characterization of a monoclonal antibody to an antigen expressed on activated macrophages. Proc Natl Acad Sci USA 81:4505–4509CrossRefGoogle Scholar
  17. Liao TL, Wu CY, Su WC, Jeng KS, Lai MM (2010) Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication. EMBO J 29:3879–3890CrossRefGoogle Scholar
  18. Liu X, Sun L, Yu M, Wang Z, Xu C, Xue Q, Zhang K, Ye X, Kitamura Y, Liu W (2009) Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication. Cell Microbiol 11:730–741CrossRefGoogle Scholar
  19. Liu X, Zhao Z, Xu C, Sun L, Chen J, Zhang L, Liu W (2012) Cyclophilin A restricts influenza A virus replication through degradation of the M1 protein. PLoS ONE 7:e31063CrossRefGoogle Scholar
  20. Liu D, Shi W, Shi Y, Wang D, Xiao H, Li W, Bi Y, Wu Y, Li X, Yan J, Liu W, Zhao G, Yang W, Wang Y, Ma J, Shu Y, Lei F, Gao GF (2013) Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet 381:1926–1932CrossRefGoogle Scholar
  21. Liu W, Li J, Zheng W, Shang Y, Zhao Z, Wang S, Bi Y, Zhang S, Xu C, Duan Z, Zhang L, Wang YL, Jiang Z, Liu W, Sun L (2017) Cyclophilin A-regulated ubiquitination is critical for RIG-I-mediated antiviral immune responses. Elife 6:e24425CrossRefGoogle Scholar
  22. Luban J (2007) Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection. J Virol 81:1054–1061CrossRefGoogle Scholar
  23. Martin K, Helenius A (1991) Nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (M1) promotes export and inhibits import. Cell 67:117–130CrossRefGoogle Scholar
  24. Rudnicka A, Yamauchi Y (2016) Ubiquitin in influenza virus entry and innate immunity. Viruses 8:293CrossRefGoogle Scholar
  25. Sha B, Luo M (1997) Structure of a bifunctional membrane-RNA binding protein, influenza virus matrix protein M1. Nat Struct Biol 4:239–244CrossRefGoogle Scholar
  26. Shimizu T, Takizawa N, Watanabe K, Nagata K, Kobayashi N (2011) Crucial role of the influenza virus NS2 (NEP) C-terminal domain in M1 binding and nuclear export of vRNP. FEBS Lett 585:41–46CrossRefGoogle Scholar
  27. Su WC, Chen YC, Tseng CH, Hsu PW, Tung KF, Jeng KS, Lai MM (2013) Pooled RNAi screen identifies ubiquitin ligase Itch as crucial for influenza A virus release from the endosome during virus entry. Proc Natl Acad Sci USA 110:17516–17521CrossRefGoogle Scholar
  28. Sun S, Guo M, Zhang JB, Ha A, Yokoyama KK, Chiu RH (2014) Cyclophilin A (CypA) interacts with NF-kappaB subunit, p65/RelA, and contributes to NF-kappaB activation signaling. PLoS ONE 9:e96211CrossRefGoogle Scholar
  29. Wang S, Zhao Z, Bi Y, Sun L, Liu X, Liu W (2013) Tyrosine 132 phosphorylation of influenza A virus M1 protein is crucial for virus replication by controlling the nuclear import of M1. J Virol 87:6182–6191CrossRefGoogle Scholar
  30. Widjaja I, de Vries E, Tscherne DM, Garcia-Sastre A, Rottier PJ, de Haan CA (2010) Inhibition of the ubiquitin–proteasome system affects influenza A virus infection at a postfusion step. J Virol 84:9625–9631CrossRefGoogle Scholar
  31. Wu CY, Jeng KS, Lai MM (2011) The SUMOylation of matrix protein M1 modulates the assembly and morphogenesis of influenza A virus. J Virol 85:6618–6628CrossRefGoogle Scholar
  32. Xu C, Meng S, Liu X, Sun L, Liu W (2010) Chicken cyclophilin A is an inhibitory factor to influenza virus replication. Virol J 7:372CrossRefGoogle Scholar
  33. Ye ZP, Pal R, Fox JW, Wagner RR (1987) Functional and antigenic domains of the matrix (M1) protein of influenza A virus. J Virol 61:239–246PubMedPubMedCentralGoogle Scholar
  34. Zhu Y, Qi X, Cui L, Zhou M, Wang H (2013) Human co-infection with novel avian influenza A H7N9 and influenza A H3N2 viruses in Jiangsu province, China. Lancet 381:2134CrossRefGoogle Scholar

Copyright information

© Wuhan Institute of Virology, CAS and Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of MicrobiologyChinese Academy of SciencesBeijingChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources and Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary MedicineGuangxi UniversityNanningChina
  4. 4.College of Life SciencesHenan Agricultural UniversityZhengzhouChina

Personalised recommendations