CypA Regulates AIP4-Mediated M1 Ubiquitination of Influenza A Virus

doi:10.1007/s12250-018-0058-6

. 2018 Oct;33(5):440-448.

doi: 10.1007/s12250-018-0058-6. Epub 2018 Oct 16.

CypA Regulates AIP4-Mediated M1 Ubiquitination of Influenza A Virus

Madina Mahesutihan^{1

2}, Weinan Zheng¹, Liang Cui^{1

2}, Yun Li¹, Pengtao Jiao³, Wenxian Yang^{1

2}, Wei Liu⁴, Jing Li¹, Wenhui Fan¹, Limin Yang¹, Wenjun Liu^{5

6}, Lei Sun^{7

8}

Affiliations

¹ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
² University of Chinese Academy of Sciences, Beijing, 100049, China.
³ State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources and Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, 530004, China.
⁴ College of Life Sciences, Henan Agricultural University, Zhengzhou, 450002, China.
⁵ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. liuwj@im.ac.cn.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China. liuwj@im.ac.cn.
⁷ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. sunlei362@im.ac.cn.
⁸ University of Chinese Academy of Sciences, Beijing, 100049, China. sunlei362@im.ac.cn.

PMID: 30328013
PMCID: PMC6235765
DOI: 10.1007/s12250-018-0058-6

CypA Regulates AIP4-Mediated M1 Ubiquitination of Influenza A Virus

Madina Mahesutihan et al. Virol Sin. 2018 Oct.

. 2018 Oct;33(5):440-448.

doi: 10.1007/s12250-018-0058-6. Epub 2018 Oct 16.

Authors

Madina Mahesutihan^{1

2}, Weinan Zheng¹, Liang Cui^{1

2}, Yun Li¹, Pengtao Jiao³, Wenxian Yang^{1

2}, Wei Liu⁴, Jing Li¹, Wenhui Fan¹, Limin Yang¹, Wenjun Liu^{5

6}, Lei Sun^{7

8}

Affiliations

¹ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.
² University of Chinese Academy of Sciences, Beijing, 100049, China.
³ State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources and Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning, 530004, China.
⁴ College of Life Sciences, Henan Agricultural University, Zhengzhou, 450002, China.
⁵ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. liuwj@im.ac.cn.
⁶ University of Chinese Academy of Sciences, Beijing, 100049, China. liuwj@im.ac.cn.
⁷ CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. sunlei362@im.ac.cn.
⁸ University of Chinese Academy of Sciences, Beijing, 100049, China. sunlei362@im.ac.cn.

PMID: 30328013
PMCID: PMC6235765
DOI: 10.1007/s12250-018-0058-6

Abstract

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.

Keywords: AIP4; Cyclophilin A (CypA); Influenza A virus (IAV); M1; Ubiquitination.

PubMed Disclaimer

Conflict of interest statement

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.

Figures

**Fig. 1**
AIP4 mediates K48-linked ubiquitination of M1. (A) Immunoblot analysis of lysates of 293T cells transfected with Flag-vector, Flag-AIP4, Flag-TRIM25, or Flag-Smurf-1, along with Myc-M1 and HA-Ub for 36 h, followed by immunoprecipitation (IP) with anti-M1 antibody. (B) Immunoblot analysis of lysates of 293T cells transfected with HA-Ub, HA-Ub-K48 or HA-Ub-K63, along with Flag-AIP4 and Myc-M1 for 36 h, followed by IP with anti-M1 antibody.

**Fig. 2**
AIP4 affects the stability of M1. (A) Immunoblot analysis of lysates of 293T cells transfected with Myc-M1 and Flag-AIP4 for 24 h, then treated with 100 µg/mL CHX, along with 10 µmol/L MG132, 10 µmol/L NH₄Cl or DMSO for 9 h (left). The relative density of M1 was normalized to β-actin (right). (B) Immunoblot analysis of lysates of 293T cells transfected with Flag-AIP4 or Flag-vector for 24 h, then infected with the influenza virus A/WSN/1933(H1N1) (MOI = 1) for 9 h (left). The relative density of M1 was normalized to β-actin (right). Data are shown as mean ± SD (n = 3). ***P < 0.001 (unpaired, two-tailed student’s t test).

**Fig. 3**
AIP4 regulates M1 ubiquitination at K102 and K104. (A) Schematic diagram representing the functional domains of M1. K21, K35, K47, K57, K95, K98, K102, K104, K113, and K187 of M1 were displayed. NEP, nuclear export protein. NLS, nuclear localization signal. (B) Immunoblot analysis of lysates of 293T cells transfected with the indicated plasmids for 36 h, followed by IP with anti-M1 antibody. (C) Immunoblot analysis of lysates in lysates of 293T cells transfected with the indicated plasmids for 36 h.

**Fig. 4**
CypA inhibits AIP4-mediated M1 ubiquitination by impairing the interaction between AIP4 and M1. (A) Immunoblot analysis of lysates of 293T cells transfected with the indicated plasmids for 36 h, followed by IP with anti-M1 antibody. (B) Immunoblot analysis of lysates of 293T/CypA-cells transfected with the indicated plasmids for 36 h, followed by IP with anti-Flag beads. (C) Immunoblot analysis of lysates in 293T/CypA- cells transfected with Myc-M1 and Flag-AIP4, along with Flag-CypA or Flag-vector for 24 h, then treated with 100 µg/mL CHX for the indicated durations (left). The relative density of M1 was normalized to β-actin (right).

**Fig. 5**
CypA regulates the cellular distribution of M1. (A) Cellular location of M1 determined by immunofluorescence assays. 293T/CypA-cells were transfected with Myc-M1 WT (WT) and Myc-M1 K102R/K104R (KK) respectively, and co-transfected with or without Flag-CypA for the indicated times. The localization of Myc-M1 (green) and Flag-CypA (red) was determined using immunofluorescence assays with anti-Myc monoclonal antibody and anti-Flag monoclonal antibody. The nuclei were stained with DAPI (blue). Scale bars, 10 μm. (B) The cellular distribution of M1 in cells. At least 100 cells from each group were scored as predominantly nuclear (N), nuclear and cytoplasmic (N + C), or predominantly cytoplasmic (C), and the percentage of cells is shown. Error bars represent the standard error of the mean from at least three independent experiments.

See this image and copyright information in PMC

Cited by

Acetylation, Methylation and Allysine Modification Profile of Viral and Host Proteins during Influenza A Virus Infection.
Ahmed F, Kleffmann T, Husain M. Ahmed F, et al. Viruses. 2021 Jul 20;13(7):1415. doi: 10.3390/v13071415. Viruses. 2021. PMID: 34372620 Free PMC article.
PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.
Hui X, Cao L, Xu T, Zhao L, Huang K, Zou Z, Ren P, Mao H, Yang Y, Gao S, Sun X, Lin X, Jin M. Hui X, et al. J Virol. 2022 Aug 10;96(15):e0078622. doi: 10.1128/jvi.00786-22. Epub 2022 Jul 21. J Virol. 2022. PMID: 35861516 Free PMC article.
PROTAC _targeting cyclophilin A controls virus-induced cytokine storm.
Li H, Yang W, Li H, Bai X, Zhang H, Fan W, Liu W, Sun L. Li H, et al. iScience. 2023 Aug 3;26(9):107535. doi: 10.1016/j.isci.2023.107535. eCollection 2023 Sep 15. iScience. 2023. PMID: 37636080 Free PMC article.
Repurposing of cyclophilin A inhibitors as broad-spectrum antiviral agents.
Han J, Lee MK, Jang Y, Cho WJ, Kim M. Han J, et al. Drug Discov Today. 2022 Jul;27(7):1895-1912. doi: 10.1016/j.drudis.2022.05.016. Epub 2022 May 21. Drug Discov Today. 2022. PMID: 35609743 Free PMC article. Review.
Phosphorylation of Influenza A Virus Matrix Protein 1 at Threonine 108 Controls Its Multimerization State and Functional Association with the STRIPAK Complex.
Liu L, Weber A, Linne U, Shehata M, Pleschka S, Kracht M, Schmitz ML. Liu L, et al. mBio. 2023 Feb 28;14(1):e0323122. doi: 10.1128/mbio.03231-22. Epub 2023 Jan 5. mBio. 2023. PMID: 36602306 Free PMC article.

See all "Cited by" articles

References

1. Arranz R, Coloma R, Chichon FJ, Conesa JJ, Carrascosa JL, Valpuesta JM, Ortin J, Martin-Benito J. The structure of native influenza virion ribonucleoproteins. Science. 2012;338:1634–1637. doi: 10.1126/science.1228172. - DOI - PubMed
1. Baudin F, Petit I, Weissenhorn W, Ruigrok RW. In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology. 2001;281:102–108. doi: 10.1006/viro.2000.0804. - DOI - PubMed
1. Bui M, Wills EG, Helenius A, Whittaker GR. Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol. 2000;74:1781–1786. doi: 10.1128/JVI.74.4.1781-1786.2000. - DOI - PMC - PubMed
1. Burleigh LM, Calder LJ, Skehel JJ, Steinhauer DA. Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J Virol. 2005;79:1262–1270. doi: 10.1128/JVI.79.2.1262-1270.2005. - DOI - PMC - PubMed
1. Calder LJ, Wasilewski S, Berriman JA, Rosenthal PB. Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci USA. 2010;107:10685–10690. doi: 10.1073/pnas.1002123107. - DOI - PMC - PubMed

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- BioCyc
- GlyGen glycoinformatics resource
Research Materials
- NCI CPTC Antibody Characterization Program

[1] Arranz R, Coloma R, Chichon FJ, Conesa JJ, Carrascosa JL, Valpuesta JM, Ortin J, Martin-Benito J. The structure of native influenza virion ribonucleoproteins. Science. 2012;338:1634–1637. doi: 10.1126/science.1228172. - DOI - PubMed

[2] Arranz R, Coloma R, Chichon FJ, Conesa JJ, Carrascosa JL, Valpuesta JM, Ortin J, Martin-Benito J. The structure of native influenza virion ribonucleoproteins. Science. 2012;338:1634–1637. doi: 10.1126/science.1228172. - DOI - PubMed

[3] Baudin F, Petit I, Weissenhorn W, Ruigrok RW. In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology. 2001;281:102–108. doi: 10.1006/viro.2000.0804. - DOI - PubMed

[4] Baudin F, Petit I, Weissenhorn W, Ruigrok RW. In vitro dissection of the membrane and RNP binding activities of influenza virus M1 protein. Virology. 2001;281:102–108. doi: 10.1006/viro.2000.0804. - DOI - PubMed

[5] Bui M, Wills EG, Helenius A, Whittaker GR. Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol. 2000;74:1781–1786. doi: 10.1128/JVI.74.4.1781-1786.2000. - DOI - PMC - PubMed

[6] Bui M, Wills EG, Helenius A, Whittaker GR. Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins. J Virol. 2000;74:1781–1786. doi: 10.1128/JVI.74.4.1781-1786.2000. - DOI - PMC - PubMed

[7] Burleigh LM, Calder LJ, Skehel JJ, Steinhauer DA. Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J Virol. 2005;79:1262–1270. doi: 10.1128/JVI.79.2.1262-1270.2005. - DOI - PMC - PubMed

[8] Burleigh LM, Calder LJ, Skehel JJ, Steinhauer DA. Influenza a viruses with mutations in the m1 helix six domain display a wide variety of morphological phenotypes. J Virol. 2005;79:1262–1270. doi: 10.1128/JVI.79.2.1262-1270.2005. - DOI - PMC - PubMed

[9] Calder LJ, Wasilewski S, Berriman JA, Rosenthal PB. Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci USA. 2010;107:10685–10690. doi: 10.1073/pnas.1002123107. - DOI - PMC - PubMed

[10] Calder LJ, Wasilewski S, Berriman JA, Rosenthal PB. Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci USA. 2010;107:10685–10690. doi: 10.1073/pnas.1002123107. - DOI - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed