Identification of host proteins that interact with African swine fever virus pE301R

Abstract

African swine fever virus (ASFV) infection poses enormous threats and challenges to the global pig industry; however, no effective vaccine is available against ASFV, attributing to the huge viral genome (approximately189 kb) and numerous encoding products (>150 genes) due to the limited understanding on the molecular mechanisms of viral pathogenesis. Elucidating the host-factor/viral-protein interaction network will reveal new targets for developing novel antiviral therapies. Using proteomic analysis, we identified 255 cellular proteins that interact with the ASFV-encoded pE301R protein when transiently expressed in HEK293T cells. Gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment, and protein-protein interaction (PPI) network analyses revealed that pE301R-interacting host proteins are potentially involved in various biological processes, including protein translation and folding, response to stimulation, and mitochondrial transmembrane transport. The interactions of two putative cellular proteins (apoptosis inducing factor mitochondria associated 1 (AIFM1) and vimentin (VIM)) with pE301R-apoptosis inducing factor have been verified by co-immunoprecipitation. Our study revealed the inhibitory role of pE301R in interferon (IFN) induction that involves VIM sequestration by pE301R, identified interactions between ASFV pE301R and cellular proteins, and predicted the potential function of pE301R and its associated biological processes, providing valuable information to enhance our understanding of viral protein function, pathogenesis, and potential candidates for the prevention and control of ASFV infection.

Keywords

African swine fever virus / pE301R protein / Protein-protein interaction network / GO and KEGG analysis / Interferon

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Given their roles as Editor-in-Chief and Associate Editor, respectively, Dr. Youming Zhang and Dr. Cheng-Ming Chiang had no involvement in the peer-review of this article, and had no access to information regarding its peer-review. Full responsibility for the editorial process for this article was delegated to Dr. Wenyuan Han.

CRediT authorship contribution statement

Menghan Shi: Software, Methodology, Formal analysis, Data curation. Niu Zhou: Writing - review & editing, Software, Methodology. Mengchen Xiu: Methodology, Investigation, Data curation. Xiangzhi Li: Writing - review & editing, Visualization, Formal analysis. Fen Shan: Writing - review & editing, Software. Wu Chen: Writing - review & editing. Wanping Li: Software, Methodology. Cheng-Ming Chiang: Writing - review & editing, Funding acquisition, Formal analysis, Data curation, Conceptualization. Xiaodong Wu: Resources, Project administration, Conceptualization. Youming Zhang: Supervision, Project administration, Funding acquisition. Aiying Li: Writing - review & editing, Visualization, Validation, Methodology. Jingjing Cao: Writing - review & editing, Writing - original draft, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization.

Acknowledgments

This work was supported by the National Key R&D Program of China (2019YFA0905700, 2018YFA0900400), Natural Science Foundation of China (31900147, 32170038, 32270088, M-0348 and 32161133013), the 111 Project (B16030), and a Sino-German Helmholtz International Lab grant. C.M.C's research was supported by US National Institutes of Health grant 1R01CA251698-01 and CPRIT grants RP180349 and RP190077.

References

[1] D.M. Zhao, E.C. Sun, L.Y. Huang, L.L. Ding, Y.M. Zhu, J.W. Zhang, D.D. Shen, X. F. Zhang, Z.J. Zhang, T. Ren, W. Wang, F. Li, X.J. He, Z.G. Bu, Highly lethal geno- type I and II recombinant African swine fever viruses detected in pigs, Nat. Commun. 14 (2023) 3096.

[2] L.K. Dixon, K. Stahl, F. Jori, L. Vial, D.U. Pfeiffer, African swine fever epidemiology and control, Annu Rev. Anim. Biosci. 8 (2020) 221-246.

[3] I. Galindo, C. Alonso, African swine fever virus: a review, Viruses 9 (2017) 103.

[4] W. Liu, H. Li, B. Liu, T. Lv, C. Yang, S. Chen, L. Feng, L. Lai, Z. Duan, X. Chen, P. Li, S. Guan, L. Chen, A new vaccination regimen using adenovirus-vectored vac- cine confers effective protection against African swine fever virus in swine, Emerg. Microbes. Infect. 12 (2023) 2233643.

[5] Y. Revilla, D. Perez-Nunez, J.A. Richt, African swine fever virus biology and vaccine approaches, Adv. Virus. Res. 100 (2018) 41-74.

[6] D. Tao, D. Sun, Y. Liu, S. Wei, Z. Yang, T. An, F. Shan, Z. Chen, J. Liu, One year of African swine fever outbreak in China, Acta Trop. 211 (2020) 105602.

[7] K. Wu, J. Liu, L. Wang, S. Fan, Z. Li, Y. Li, L. Yi, H. Ding, M. Zhao, J. Chen, Current state of global African swine fever vaccine development under the prevalence and transmission of ASF in China, Vaccines. (Basel) 8 (2020).

[8] L.K. Dixon, D.A. Chapman, C.L. Netherton, C. Upton, African swine fever virus repli- cation and genomics, Virus. Res. 173 (2013) 3-14.

[9] N.N. Gaudreault, D.W. Madden, W.C. Wilson, J.D. Trujillo, J.A. Richt, African swine fever virus: an emerging DNA arbovirus, Front. Vet. Sci. 7 (2020) 215.

[10] A. Karger, D. Perez-Nunez, J. Urquiza, P. Hinojar, C. Alonso, F.B. Freitas, Y. Revilla, M. F. Le Potier, M. Montoya, An update on african swine fever virology, Viruses 11 (2019) 864.

[11] A.L. Munoz, E. Tabares, Characteristics of the major structural proteins of African swine fever virus: role as antigens in the induction of neutralizing antibodies. A review, Virology 571 (2022) 46-51.

[12] P.P. Zhou, L.F. Li, K.H. Zhang, B. Wang, L.J. Tang, M. Li, T. Wang, Y. Sun, S. Li, H.J. Qiu, Deletion of the gene of African swine fever virus decreases infectious progeny virus production due to aberrant virion morphogenesis and enhances in- flammatory cytokine expression in porcine macrophages (vol 96, e01667-21, 2021),

[13] Y. Wang, W. Kang, W. Yang, J. Zhang, D. Li, H. Zheng, Structure of African swine fever virus and associated molecular mechanisms underlying infection and immuno- suppression: a review, Front. Immunol. 12 (2021) 715582.

[14] C.L. Afonso, M.E. Piccone, K.M. Zaffuto, J. Neilan, G.F. Kutish, Z. Lu, C.A. Balinsky, T.R. Gibb, T.J. Bean, L. Zsak, D.L. Rock, African swine fever virus multigene family 360 and 530 genes affect host interferon response, J. Virol. 78 (2004) 1858-1864.

[15] M. Ding, W. Dang, H. Liu, F. Xu, H. Huang, Y. Sunkang, T. Li, J. Pei, X. Liu, Y. Zhang, H. Zheng, Combinational deletions of MGF360- 9L and MGF505-7R at- tenuated highly virulent African swine fever virus and conferred protection against

[16] Y.Y. Qi Gao, Y. Luo, X. Chen, T. Gong, D. Wu, Y. Feng, X. Zheng, H. Wang, G. Zhang, G. Lu, L. Gong, African swine fever virus envelope glycoprotein CD2v interacts with host CSF2RA to regulate the JAK2-STAT3 pathway and inhibit apoptosis to facilitate virus replication, J. Virol. 97 (2024) e0188922.

[17] L. Yu, Z. Zhu, J. Deng, K. Tian, X. Li, Antagonisms of ASFV towards host defense mechanisms: knowledge gaps in viral immune evasion and pathogenesis, Viruses 15 (2023) 574.

[18] S. Correia, S. Ventura, R.M. Parkhouse, Identification and utility of innate immune system evasion mechanisms of ASFV, Virus. Res. 173 (2013) 87-100.

[19] L.K. Dixon, T. Wang, R. Luo, J. Zhang, Z. Lu, L.F. Li, Y.H. Zheng, L. Pan, J. Lan, H. Zhai, S. Huang, Y. Sun, H.J. Qiu, The MGF300-2 R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degrada- tion of IKK 𝛼 and IKK𝛽 through the recruitment of TOLLIP, PLoS. Pathog. 19 (2023) e1011580.

[20] P. Zhou, J. Dai, K. Zhang, T. Wang, L.F. Li, Y. Luo, Y. Sun, H.J. Qiu, S. Li, The H240R protein of African swine fever virus inhibits interleukin 1beta production by inhibit- ing NEMO expression and NLRP3 oligomerization, J. Virol. 96 (2022) e0095422.

[21] A. Ayanwale, S. Trapp, R. Guabiraba, I. Caballero, F. Roesch, New insights in the interplay between african swine fever virus and innate immunity and its impact on viral pathogenicity, Front. Microbiol. 13 (2022) 958307.

[22] A.E. Afe, Z.J. Shen, X. Guo, R. Zhou, K. Li, African swine fever virus interaction with host innate immune factors, Viruses 15 (2023) 1220.

[23] F. Zhang, A. Moon, K. Childs, S. Goodbourn, L.K. Dixon, The African swine fever virus DP71L protein recruits the protein phosphatase 1 catalytic subunit to dephosphory- late eIF2alpha and inhibits CHOP induction but is dispensable for these activities during virus infection, J. Virol. 84 (2010) 10681-10689.

[24] L.K. Dixon, P.J. Sanchez-Cordon, I. Galindo, C. Alonso, Investigations of pro- and an- ti-apoptotic factors affecting African swine fever virus replication and pathogenesis, Viruses. 9 (2017).

[25] Z. Shen, C. Chen, Y. Yang, Z. Xie, Q. Ao, L. Lv, S. Zhang, H. Chen, R. Hu, H. Chen, G. Peng, A novel function of African Swine Fever Virus pE66L in inhibition of host translation by the PKR/eIF2alpha pathway, J. Virol. 95 (2021).

[26] C. Hurtado, A.G. Granja, M.J. Bustos, M.L. Nogal, G.G. de Buitrago, V.G. de Yebenes, M. L. Salas, Y. Revilla, A.L. Carrascosa, The C-type lectin homologue gene (EP153R) of African swine fever virus inhibits apoptosis both in virus infection and in heterol- ogous expression, Virology. 326 (2004) 160-170.

[27] M.V. Borca, E. Ramirez-Medina, E. Silva, E. Vuono, A. Rai, S. Pruitt, L.G. Holinka, L. Velazquez-Salinas, J. Zhu, D.P. Gladue, Development of a highly effective African swine fever virus vaccine by deletion of the I177L gene results in sterile immunity against the current epidemic eurasia strain, J. Virol. 94 (2020).

[28] A.L. Reis, L.C. Goatley, T. Jabbar, P.J. Sanchez-Cordon, C.L. Netherton, D.A.G. Chap- man, L.K. Dixon, Deletion of the African swine fever virus gene DP148R does not reduce virus replication in culture but reduces virus virulence in pigs and induces high levels of protection against challenge, J. Virol. 91 (2017) e01428-17.

[29] A. Crespo-Bellido, S. Duffy, The how of counter-defense: viral evolution to combat host immunity, Curr. Opin. Microbiol. 74 (2023) 102320.

[30] N.C. Asensio, E.M. Giner, N.S. de Groot, M.T. Burgas, Centrality in the host-pathogen interactome is associated with pathogen fitness during infection, Nat. Commun. 8 (2017) 14092.

[31] J. Zhou, Y. Wang, L. Zhou, Y. Qiu, J. Zhao, B. Dai, X. Feng, L. Hou, J. Liu, Interaction network of porcine circovirus type 3 and 4 capsids with host proteins, Viruses 14 (2022) 939.

[32] B. Ding, L. Zhang, Z. Li, Y. Zhong, Q. Tang, Y. Qin, M. Chen, The matrix protein of human parainfluenza virus type 3 induces mitophagy that suppresses interferon responses, Cell Host. Microbe 21 (2017) 538-547 e4.

[33] B. Yang, D. Zhang, X. Shi, C. Shen, Y. Hao, T. Zhang, J. Yang, X. Yuan, X. Chen, D. Zhao, H. Cui, D. Li, Z. Zhu, H. Tian, F. Yang, H. Zheng, K. Zhang, X. Liu, Con- struction, identification and analysis of the interaction network of african swine fever virus MGF360-9L with host proteins, Viruses 13 (2021).

[34] R.J. Yanez, J.M. Rodriguez, M.L. Nogal, L. Yuste, C. Enriquez, J.F. Rodriguez, E. Vin- uela, Analysis of the complete nucleotide sequence of African swine fever virus, Vi- rology 208 (1995) 249-278.

[35] M. O’Donnell, D. Jeruzalmi, J. Kuriyan, Clamp loader structure predicts the architec- ture of DNA polymerase III holoenzyme and RFC, Curr. Biol. 11 (2001) R935-R946.

[36] J. Wu, H. Zheng, P. Gong, Crystal structure of African swine fever virus pE301R reveals a ring-shaped trimeric DNA sliding clamp, J. Biol. Chem. 299 (2023) 104872.

[37] S. Li, H.L. Ge, Y.H. Li, K.H. Zhang, S.X. Yu, H.W. Cao, Y.J. Wang, H. Deng, J.Q. Li, J.W. Dai, L.F. Li, Y.Z. Luo, Y. Sun, Z. Geng, Y.H. Dong, H. Zhang, and H.J. Qiu, The E301R protein of African swine fever virus functions as a sliding clamp involved in viral genome replication. mBio (2023).

[38] X. Liu, H. Liu, G. Ye, M. Xue, H. Yu, C. Feng, Q. Zhou, X. Liu, L. Zhang, S. Jiao, C. Weng, L. Huang, African swine fever virus pE301R negatively regulates cGAS-ST- ING signaling pathway by inhibiting the nuclear translocation of IRF3, Vet. Micro- biol. 274 (2022) 109556.

[39] H. Liu, G. Ye, X. Liu, M. Xue, Q. Zhou, L. Zhang, K. Zhang, L. Huang, C. Weng, Vi- mentin inhibits type I interferon production by disrupting the TBK1-IKKepsilon-IRF3 axis, Cell Rep. 41 (2022) 111469.

[40] J.Y. Changjie Lv, Li Zhao, C. Wu, C. Kang, Q. Zhang, X. Sun, Xi Chen, Z. Zou, M. Jin, Infection characteristics and transcriptomics of African swine fever virus in bama minipigs. 10.1128/spectrum.03834-22 (2022).

[41] M. Kikkert, Innate immune evasion by human respiratory RNA viruses, J. Innate Immun. 12 (2020) 4-20.

[42] H. Yang, Y. Dong, Y. Bian, N. Xu, Y. Wu, F. Yang, Y. Du, T. Qin, S. Chen, D. Peng, X. Liu, The influenza virus PB 2 protein evades antiviral innate immunity by inhibit- ing JAK1/STAT signalling, Nat. Commun. 13 (2022) 6288.

[43] D.C. Beachboard, S.M. Horner, Innate immune evasion strategies of DNA and RNA viruses, Curr. Opin. Microbiol. 32 (2016) 113-119.

[44] X. Zhang, Q. Lan, M. Zhang, F. Wang, K. Shi, X. Li, E. Kuang, Inhibition of AIM2 inflammasome activation by SOX/ORF37 promotes lytic replication of Kaposi’s sar- coma-associated herpesvirus, Proc. Natl. Acad. Sci. U S A 120 (2023) e2300204120.

[45] A. Castello, A. Quintas, E.G. Sanchez, P. Sabina, M. Nogal, L. Carrasco, Y. Re- villa, Regulation of host translational machinery by African swine fever virus, PLoS Pathog. 5 (2009) e1000562.

[46] H. Zhong, S. Fan, Y. Du, Y. Zhang, A. Zhang, D. Jiang, S. Han, B. Wan, G. Zhang, African swine fever virus MGF110-7L induces host cell translation suppression and stress granule formation by activating the PERK/PKR-eIF2alpha pathway, Microbiol. Spectr. 10 (2022) e0328222.

[47] M.T. Vo, C.Y. Choi, Y.B. Choi, The mitophagy receptor NIX induces vIRF-1 oligomer- ization and interaction with GABARAPL1 for the promotion of HHV-8 reactiva- tion-induced mitophagy, PLoS Pathog. 19 (2023) e1011548.

[48] B. Hu, G. Zhong, S. Ding, K. Xu, X. Peng, W. Dong, J. Zhou, African swine fever virus protein p17 promotes mitophagy by facilitating the interaction of SQSTM1 with TOMM70, Virulence 14 (2023) 2232707.

[49] S.L. Archer, A. Dasgupta, K.H. Chen, D. Wu, K. Baid, J.E. Mamatis, V. Gonzalez, A. Read, R.E. Bentley, A.Y. Martin, J.D. Mewburn, K.J. Dunham-Snary, G.A. Evans, G. Levy, O. Jones, R. Al-Qazazi, B. Ring, E. Alizadeh, C.C. Hindmarch, J. Rossi, P.D. Lima, D. Falzarano, A. Banerjee, C.C. Colpitts, SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia, Redox. Biol. 58 (2022) 102508.

[50] S. Laukoter, H. Rauschka, A.R. Troscher, U. Kock, E. Saji, K. Jellinger, H. Lass- mann, J. Bauer, Differences in T cell cytotoxicity and cell death mechanisms between progressive multifocal leukoencephalopathy, herpes simplex virus en- cephalitis and cytomegalovirus encephalitis, Acta Neuropathol. 133 (2017) 613-627.

[51] G.Y. Zhou, B. Roizman, Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome but not the translocation of mitochondrial apopto- sis-inducing factor to the nuclei of human embryonic lung fibroblasts, J. Virol. 74 (2000) 9048-9053.

[52] X.Y. Qu, X.R. Ding, M. Duan, J. Yang, R.X. Lin, Z. Zhou, S.Q. Wang, Influenza virus infection induces translocation of apoptosis-inducing factor (AIF) in A549 cells: role of AIF in apoptosis and viral propagation, Arch. Virol. 162 (2017) 669-675.

[53] Y. Kim, C. Lee, Porcine epidemic diarrhea virus induces caspase-independent apop- tosis through activation of mitochondrial apoptosis-inducing factor, Virology 460 (2014) 180-193.

[54] J. Arrindell, B. Desnues, Vimentin: from a cytoskeletal protein to a critical modulator of immune response and a target for infection, Front. Immunol. 14 (2023).

[55] I. Ramos, K. Stamatakis, C.L. Oeste, D. Pérez-Sala, Vimentin as a multifaceted player and potential therapeutic target in viral infections, Int. J. Mol. Sci. 21 (2020).

[56] P. Deptula, K. Fiedoruk, M. Wasilewska, L. Suprewicz, M. Ciesluk, P. Zeliszewska, M. Ocwieja, Z. Adamczyk, K. Pogoda, R. Bucki, Physicochemical nature of SARS-CoV-2 spike protein binding to human vimentin, ACS. Appl. Mater. Interfaces. 15 (2023) 34172-34180.

[57] Y. Cheng, J.X. Lou, Y.Y. Liu, C.C. Liu, J. Chen, M.C. Yang, Y.B. Ye, Y.Y. Go, B. Zhou, Intracellular vimentin regulates the formation of classical swine fever virus replica- tion complex through interaction with NS5A protein, J. Virol. 97 (2023) e0177022.

[58] Y. Zhang, S. Zhao, Y. Li, F. Feng, M. Li, Y. Xue, J. Cui, T. Xu, X. Jin, Y. Jiu, Host cytoskeletal vimentin serves as a structural organizer and an RNA-binding protein regulator to facilitate Zika viral replication, Proc.Natl. Acad. Sci. U S A 119 (2022).

[59] D.P. Gladue, V. O’Donnell, R. Baker-Branstetter, L.G. Holinka, J.M. Pacheco, I. F. Sainz, Z. Lu, X. Ambroggio, L. Rodriguez, M.V. Borca, Foot-and-Mouth Disease Virus Modulates Cellular Vimentin for Virus Survival (vol 87, pg 6794, 2013), J. Virol. 93 (2019).

[60] X.Q. Ma, Y. Ling, P.H. Li, P. Sun, Y.M. Cao, X.W. Bai, K. Li, Y.F. Fu, J. Zhang, D. Li, H.F. Bao, Y.L. Chen, Z.Y. Li, Y.G. Wang, Z.J. Lu, Z.X. Liu, Cellular vimentin interacts with foot-and-mouth disease virus nonstructural protein 3a and negatively modu- lates viral replication, J. Virol. 94 (2020).

[61] X. Lu, K. Liu, Y. Chen, R. Gao, Z. Hu, J. Hu, M. Gu, S. Hu, C. Ding, X. Jiao, X. Wang, X. Liu, X. Liu, Cellular vlular vimentin regulates the infectivity of Newcastle disease virus through targeting of the HN protein, Vet, Res. 54 (2023) 92.