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Citation: Fang-Fang Jiang, Ren-Qi Wang, Chao-Yue Guo, Ke Zheng, Hai-Long Liu, Le Su, Sheng-Song Xie, Huan-Chun Chen, Zheng-Fei Liu. Phospho-proteomics identifies a critical role of ATF2 in pseudorabies virus replication [J].VIROLOGICA SINICA, 2022, 37(4) : 591-600.  http://dx.doi.org/10.1016/j.virs.2022.06.003

Phospho-proteomics identifies a critical role of ATF2 in pseudorabies virus replication

  • Corresponding author: Zheng-Fei Liu, lzf6789@mail.hzau.edu.cn
  • Received Date: 22 July 2021
    Accepted Date: 02 June 2022
    Available online: 07 June 2022
  • Pseudorabies virus (PRV), an etiological agent of pseudorabies in livestock, has negatively affected the porcine industry all over the world. Epithelial cells are reported as the first site of PRV infection. However, the role of host proteins and its related signaling pathways in PRV replication is largely unclear. In this study, we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney (PK-15) epithelial cells. Totally 5723 phosphopeptides, corresponding to 2180 proteins, were obtained, and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells (P < 0.05). GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways. Further functional studies of NF-κB, transcription activator factor-2 (ATF2), MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference (RNAi) knockdown. It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number. JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV, especially during the biogenesis of viral genome DNA. Subsequently, by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2, it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication. These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.

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    1. An, T.Q., Peng, J.M., Tian, Z.J., Zhao, H.Y., Li, N., Liu, Y.M., Chen, J.Z., Leng, C.L., Sun, Y., Chang, D., Tong, G.Z., 2013. Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012. Emerg Infect Dis 19, 1749–1755.

    2. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., 2000. Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25.

    3. Beausoleil, S.A., Villén, J., Gerber, S.A., Rush, J., Gygi, S.P., 2006. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.Nature biotechnology 24, 1285.

    4. Beck, F., Geiger, J., Gambaryan, S., Solari, F.A., Dell'Aica, M., Loroch, S., Mattheij, N.J., Mindukshev, I., Pötz, O., Jurk, K., Burkhart, J.M., Fufezan, C., Heemskerk, J.W., Walter, U., Zahedi, R.P., Sickmann, A., 2017. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Blood 129, e1–e12.

    5. Bhoumik, A., Ronai, Z., 2008. ATF2-A transcription factor that elicits oncogenic or tumor suppressor activities. Cell Cycle 7, 2341–2345.

    6. Duzgun, S.A., Yerlikaya, A., Zeren, S., Bayhan, Z., Okur, E., Boyaci, I., 2017. Differential effects of p38 MAP kinase inhibitors SB203580 and SB202190 on growth and migration of human MDA-MB-231 cancer cell line. Cytotechnology 69, 711–724.

    7. Flori, L., Rogel-Gaillard, C., Cochet, M., Lemonnier, G., Hugot, K., Chardon, P., Robin, S., Lefevre, F., 2008. Transcriptomic analysis of the dialogue between Pseudorabies virus and porcine epithelial cells during infection. BMC Genom. 9, 123.

    8. Freuling, C.M., Muller, T.F., Mettenleiter, T.C., 2017. Vaccines against pseudorabies virus(PrV). Vet. Microbiol. 206, 3–9.

    9. Gueorguiev, V.D., Cheng, S.Y., Sabban, E.L., 2006. Prolonged activation of cAMPresponse element-binding protein and ATF-2 needed for nicotine-triggered elevation of tyrosine hydroxylase gene transcription in PC12 cells. J. Biol. Chem. 281, 10188–10195.

    10. Gupta, S., Campbell, D., Derijard, B., Davis, R.J., 1995. Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267, 389–393.

    11. Hai, T.W., Liu, F., Coukos, W.J., Green, M.R., 1989. Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNAbinding heterodimers. Genes Dev. 3, 2083–2090.

    12. He, F., Xiao, Z., Yao, H., Li, S., Feng, M., Wang, W., Liu, Z., Wu, J., 2019. The protective role of microrna-21 against coxsackievirus b3 infection through targeting the map2k3/p38 mapk signaling pathway. J. Transl. Med. 17, 335.

    13. Inoue, S., Mizushima, T., Ide, H., Jiang, G., Goto, T., Nagata, Y., Netto, G.J., Miyamoto, H., 2018. ATF2 promotes urothelial cancer outgrowth via cooperation with androgen receptor signaling. Endocr. Connect 7, 1397–1408.

    14. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30.

    15. Kumar, A., Manna, S.K., Dhawan, S., Aggarwal, B.B., 1998. HIV-tat protein activates c-Jun N-terminal kinase and activator protein-1. J. Immunol. 161, 776–781.

    16. Larsen, M.R., Thingholm, T.E., Jensen, O.N., Roepstorff, P., Jørgensen, T.J.D., 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using Titanium dioxide microcolumns. Mol. Cell. Proteomics 4, 873–886.

    17. Li, A., Lu,G., Qi,J., Wu, L., Tian, K.,Luo, T., Shi, Y., Yan, J., Gao, G.F., 2017. Structural basis of nectin-1 recognition by pseudorabies virus glycoprotein D. PLoS Pathog. 13, e1006314.

    18. Lim, J.Y., Park, S.J., Hwang, H.Y., Park, E.J., Nam, J.H., Kim, J., Park, S.I., 2005. TGF-beta 1 induces cardiac hypertrophic responses via PKC-dependent ATF-2 activation. J. Mol. Cell. Cardiol. 39, 627–636.

    19. Liu, F., Zheng, H., Tong, W., Li, G.X., Tian, Q., Liang, C., Li, L.W., Zheng, X.C., Tong, G.Z., 2016. Identification and analysis of novel viral and host dysregulated micrornas in variant pseudorabies virus-infected pk15 cells. PLoS One 11, e0151546.

    20. Ma, J., Chang, K., Peng, J., Shi, Q., Gan, H., Gao, K., Feng, K., Xu, F., Zhang, H., Dai, B., Zhu, Y., Shi, G., Shen, Y., Zhu, Y., Qin, X., Li, Y., Zhang, P., Ye, D., Wang, C., 2018. Spop promotes atf2 ubiquitination and degradation to suppress prostate cancer progression. J. Exp. Clin. Cancer Res. 37, 145.

    21. Macek, B., Mann, M., Olsen, J.V., 2009. Global and site-specific quantitative phosphoproteomics: principles and applications. Annu. Rev. Pharmacol. Toxicol. 49, 199–221.

    22. McLean, T., Bachenheimer, S., 1999. Activation of cjun n-terminal kinase by herpes simplex virus type 1 enhances viral replication. J. Virol. 73, 8415–8426.

    23. Mettenleiter, T.C., 2000. Aujeszky's disease (pseudorabies) virus: the virus and molecular pathogenesis-state of the art. Vet. Res. 31, 99–115.

    24. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.

    25. Moynagh, J., 1997. Aujeszky's disease and the European Community. Vet. Microbiol. 55, 159–166.

    26. Murata, T., Noda, C., Saito, S., Kawashima, D., Sugimoto, A., Isomura, H., Kanda, T., Yokoyama, K.K., Tsurumi, T., 2011. Involvement of jun dimerization protein 2 (jdp2) in the maintenance of epstein-barr virus latency. J. Bio. Chem. 286, 22007–22016.

    27. Olsen, J.V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., Mann, M., 2006. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648.

    28. Ouwens, D.M., de Ruiter, N.D., van der Zon, G.C., Carter, A.P., Schouten, J., van der Burgt, C., Kooistra, K., Bos, J.L., Maassen, J.A., van Dam, H., 2002. Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38. Embo J. 21, 3782–3793.

    29. Pensaert, M., Morrison, R., 2000. Challenges of the final stages of the adv eradication program. Vet. Res. 31, 141–145.

    30. Ravikumar, V., Jers, C., Mijakovic, I., 2015. Elucidating host-pathogen interactions based on post-translational mmodifications using proteomics aapproaches. Front. Microbiol. 6, 1313.

    31. Söderholm, S., Kainov, D.E., Öhman, T., Denisova, O.V., Schepens, B., Kulesskiy, E., Imanishi, S.Y., Corthals, G., Hintsanen, P., Aittokallio, T., 2016. Phosphoproteomics to characterize host response during influenza A virus infection of human macrophages. Mol. Cell. Proteomics 15, 3203–3219.

    32. Recio, J.A., Merlino, G., 2002. Hepatocyte growth factor/scatter factor activates proliferation in melanoma cells through p38 MAPK, ATF-2 and cyclin D1. Oncogene 21, 1000–1008.

    33. Rodems, S.M., Spector, D.H., 1998. Extracellular signal-regulated kinase activity is sustained early during human cytomegalovirus infection. J. Virol. 72, 9173–9180.

    34. Salinas-Abarca, A.B., Velazquez-Lagunas, I., Franco-Enzástiga, Ú., Torres-López, J.E., Rocha-González, H.I., Granados-Soto, V., 2018. ATF2, but not ATF3, participates in the maintenance of nerve injury-induced tactile allodynia and thermal hyperalgesia. Mol. Pain 9, 12.

    35. Schagger, H., 2006. Tricine-SDS-PAGE. Nat. Protocols 1, 16–22.

    36. Sharma-Walia, N., Krishnan, H.H., Naranatt, P.P., Zeng, L., Smith, M.S., Chandran, B., 2005. ERK1/2 and mek1/2 induced by kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) early during infection of target cells are essential for expression of viral genes and for establishment of infection. J. Virol. 79, 10308–10329.

    37. Shen, H., Wu, N., Wang, Y., Han, X., Zheng, Q., Cai, X., Zhang, H., Zhao, M., 2017. JNK inhibitor SP600125 aparaquat-induced alung injury: an in vivo and in vitro study. Inflammation 40, 1319–1330.

    38. Stevens, J.G., Wagner, E., Devi-Rao, G., Cook, M., Feldman, L., 1987. RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons. Science 235, 1056–1059.

    39. Tombacz, D., Toth, J.S., Boldogkoi, Z., 2011. Deletion of the virion host shut: off gene of pseudorabies virus results in selective upregulation of the expression of early viral genes in the late stage of infection. Genomics 98, 15–25.

    40. van Dam, H., Wilhelm, D., Herr, I., Steffen, A., Herrlich, P., Angel, P., 1995. ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J. 14, 1798–1811.

    41. Wang, L., Payton, R., Dai, W., Lu, L., 2011. Hyperosmotic Stress-induced ATF-2 Activation through Polo-like Kinase 3 in Human Corneal Epithelial Cells. J. Biol. Chem. 286, 1951–1958.

    42. Wang, X., Wu, C.-X., Song, X.-R., Chen, H.-C., Liu, Z.-F., 2017. Comparison of pseudorabies virus China reference strain with emerging variants reveals independent virus evolution within specific geographic regions. Virology 506, 92–98.

    43. Wang, X., Zhang, M.-M., Yan, K., Tang, Q., Wu, Y.-Q., He, W.-B., Chen, H.-C., Liu, Z.-F., 2018. The full-length microRNA cluster in the intron of large latency transcript is associated with the virulence of pseudorabies virus. Virology 520, 59–66.

    44. Wojcechowskyj, J.A., Didigu, C.A., Lee, J.Y., Parrish, N.F., Sinha, R., Hahn, B.H., Bushman, F.D., Jensen, S.T., Seeholzer, S.H., Doms, R.W., 2013. Quantitative phosphoproteomics reveals extensive cellular reprogramming during HIV-1 entry. Cell Host Microbe 13, 613–623.

    45. Wu, Y.Q., Chen, D.J., He, H.B., Chen, D.S., Chen, L.L., Chen, H.C., Liu, Z.F., 2012. Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs. PLoS One 7, e30988.

    46. Xie, S., Shen, B., Zhang, C., Huang, X., Zhang, Y., 2014. sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS One 9, e100448.

    47. Xing, J., Liang, J., Liu, S., Huang, L., Hu, P., Liu, L., Liao, M., Qi, W., 2021. Japanese encephalitis virus restricts hmgb1 expression to maintain mapk pathway activation for viral replication. Vet. Microbiol. 262, 109237.

    48. Yan, K., Liu, J., Guan, X., Yin, Y.-X., Peng, H., Chen, H.-C., Liu, Z.-F., 2019. The ctterminus of ttegument protein pUL 21 contributes to pseudorabies virus neuroinvasion. J. Virol. 93 e02052-2018.

    49. Yang, S., Pei, Y., Zhao, A., 2017. iTRAQ-based proteomic analysis of porcine kidney epithelial PK15 cells infected with pseudorabies virus. Sci. Rep. 7, 45922.

    50. Yeh, C.J., Lin, P.Y., Liao, M.H., Liu, H.J., Lee, J.W., Chiu, S.J., Hsu, H.Y., Shih, W.L., 2008. TNF-alpha mediates pseudorabies virus-induced apoptosis via the activation of p38 MAPK and JNK/SAPK signaling. Virology 381, 55–66.

    51. Yin, Y., Romero, N., Favoreel, H.W., 2021. Pseudorabies virus inhibits type I and type III interferon-induced signaling via proteasomal degradation of janus kinases. J. Virol. 95, e0079321.

    52. Yu, X.L., Zhou, Z., Hu, D., Zhang, Q., Han, T., Li, X., Gu, X., Yuan, L., Zhang, S., Wang, B., 2014. Pathogenic pseudorabies virus, china, 2012. Emerg. Infect. Dis. 20, 102–104.

    53. Yuan, Z., Gong, S., Luo, J., Zheng, Z., Song, B., Ma, S., Guo, J., Hu, C., Thiel, G., Vinson, C., Hu, C.D., Wang, Y., Li, M., 2009. Opposing roles for ATF2 and c-Fos in c-Junmediated neuronal apoptosis. Mol. Cell Biol. 29, 2431–2442.

    54. Yuan, J, Liu, X., Wu, A.W., McGonagill, P.W., Keller, M.J., Galle, C.S., Meier, J.L., 2009. Breaking human cytomegalovirus major immediate-early gene silence by vasoactive intestinal peptide stimulation of the protein kinase a-creb-torc2 signaling cascade in human pluripotent embryonal ntera2 cells. J. Virol. 83, 6391–6403.

    55. Zachos, G., Clements, B., Conner, J., 1999. Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1. J. Biol. Chem. 274, 5097–5103.

    56. Zhu, Z.X., Li, W.W., Zhang, X.L., Wang, C.C., Gao, L.L., Yang, F., Cao, W.J., Li, K.L., Tian, H., Liu, X.T., Zhang, K.S., Zheng, H.X., 2020. Foot-and-mouth disease virus capsid protein vp1 interacts with host ribosomal protein sa to maintain activation of the mapk signal pathway and promote virus replication. J. Virol. 94, e01350-19.

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    Phospho-proteomics identifies a critical role of ATF2 in pseudorabies virus replication

      Corresponding author: Zheng-Fei Liu, lzf6789@mail.hzau.edu.cn
    • a State Key Laboratory of Agricultural Microbiology, Hongshan Laboratory and Key laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China;
    • b Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China

    Abstract: Pseudorabies virus (PRV), an etiological agent of pseudorabies in livestock, has negatively affected the porcine industry all over the world. Epithelial cells are reported as the first site of PRV infection. However, the role of host proteins and its related signaling pathways in PRV replication is largely unclear. In this study, we performed a quantitative phosphoproteomics screening on PRV-infected porcine kidney (PK-15) epithelial cells. Totally 5723 phosphopeptides, corresponding to 2180 proteins, were obtained, and the phosphorylated states of 810 proteins were significantly different in PRV-infected cells compared with mock-infected cells (P < 0.05). GO and KEGG analysis revealed that these differentially expressed phosphorylated proteins were predominantly related to RNA transport and MAPK signaling pathways. Further functional studies of NF-κB, transcription activator factor-2 (ATF2), MAX and SOS genes in MAPK signaling pathway were analyzed using RNA interference (RNAi) knockdown. It showed that only ATF2-knockdown reduces both PRV titer and viral genome copy number. JNK pathway inhibition and CRISPR/Cas9 gene knockout showed that ATF2 was required for the effective replication of PRV, especially during the biogenesis of viral genome DNA. Subsequently, by overexpression of the ATF2 gene and point mutation of the amino acid positions 69/71 of ATF2, it was further demonstrated that the phosphorylation of ATF2 promoted PRV replication. These findings suggest that ATF2 may provide potential therapeutic target for inhibiting PRV infection.

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