Equine ANP32 proteins support influenza A virus RNA polymerase activity.
Abstract: Host ANP32 family proteins are crucial for maintaining the activity of influenza RNA polymerase and play an important role in the cross-species transmission of influenza viruses. To date, the molecular properties of equine ANP32 (eqANP32) protein are poorly understood, particularly the mechanisms that affect equine influenza virus (EIV) RNA polymerase activity. Here, we found that there are six alternative splicing variants of equine ANP32A (eqANP32A) with different levels of expression. Further studies showed that these six splicing variants of eqANP32A supported the activity of EIV RNA polymerase to varying degrees, with the variant eqANP32A_X2 having the highest expression abundance and exhibiting the highest support of polymerase activity. Sequence analysis demonstrated that the differences in the N-Cap regions of the six splicing variants significantly affected their N-terminal conformation, but did not affect their ability to bind RNA polymerase. We also demonstrated that there is only one transcript of eqANP32B, and that this transcript showed only very low support to the EIV RNA polymerase. This functional defect in eqANP32B is caused by the sequence of the 110-259 amino acids at its C-terminus. Our results indicated that it is the eqANP32A_X2 protein that mainly determines the efficiency of the EIV replication in horses. In conclusion, our study parsed the molecular properties of eqANP32 family proteins and revealed the sequence features of eqANP32A and eqANP32B, suggesting for the first time that the N-cap region of ANP32A protein also plays an important role in supporting the activity of the influenza virus polymerase.
Copyright © 2023 The Authors. Publishing services by Elsevier B.V. All rights reserved.
Publication Date: 2023-10-27 PubMed ID: 39491182PubMed Central: PMC10786659DOI: 10.1016/j.virs.2023.10.009Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
Equine ANP32 proteins, particularly a specific splicing variant of ANP32A, support the activity of influenza A virus RNA polymerase, influencing the efficiency of equine influenza virus replication. This study identifies different splicing variants of equine ANP32A and characterizes their impact on viral polymerase activity and cross-species viral adaptation.
Background and Importance
- ANP32 family proteins in hosts are essential for influenza virus RNA polymerase function.
- These proteins influence how influenza viruses cross species barriers and replicate efficiently.
- Equine ANP32 (eqANP32) proteins had not been thoroughly characterized prior to this study, especially regarding their support of equine influenza virus (EIV) RNA polymerase activity.
Key Findings: Equine ANP32A Variants
- Identification of six alternatively spliced variants of equine ANP32A (eqANP32A), each expressed at different levels in horse cells.
- These variants exhibit distinct abilities to support EIV RNA polymerase activity, indicating functional diversity.
- The variant named eqANP32A_X2:
- Is the most abundantly expressed among the six variants.
- Provides the strongest support for the activity of EIV RNA polymerase.
- Sequence analysis reveals that differences in the N-terminal cap (N-Cap) regions among variants change the protein’s conformation.
- Despite conformational changes, the variants maintain their capacity to bind viral RNA polymerase, meaning binding alone does not fully explain functional differences.
Equine ANP32B Characteristics
- A single transcript of eqANP32B was identified in horse cells.
- eqANP32B shows a very low ability to support EIV RNA polymerase, indicating it plays a minor role in viral replication support compared to eqANP32A variants.
- The poor support function is traced to a specific sequence within amino acids 110-259 at the C-terminal region of eqANP32B.
Implications for Viral Replication and Species Adaptation
- The eqANP32A_X2 splicing variant is concluded to be the main ANP32 protein influencing the efficiency of equine influenza virus replication in horses.
- This highlights the importance of specific host ANP32 protein variants in supporting viral life cycle activities.
- The study suggests that structural differences, especially in the N-Cap region of ANP32A, are critical in determining the extent of support to influenza polymerase activity.
- This is a novel insight since previous research emphasized mainly the ANP32 protein’s ability to bind polymerase, whereas this study points to the functional role of the N-Cap region conformation.
Conclusion and Novel Contributions
- The research parsed the molecular features of equine ANP32 proteins and determined their varying capacities to support influenza virus RNA polymerase.
- It identified eqANP32A_X2 as the primary factor driving equine influenza virus replication efficiency in its natural host.
- For the first time, it demonstrated that the N-Cap region of ANP32A influences influenza polymerase activity beyond polymerase binding alone.
- These findings deepen understanding of host factors in influenza virus adaptation and may inform future studies on cross-species infection mechanisms and antiviral targets.
Cite This Article
APA
Zhang Y, Guo X, Yu M, Sun L, Qu Y, Guo K, Hu Z, Liu D, Zhang H, Wang X.
(2023).
Equine ANP32 proteins support influenza A virus RNA polymerase activity.
Virol Sin, 38(6), 951-960.
https://doi.org/10.1016/j.virs.2023.10.009 Publication
Researcher Affiliations
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, Institute of Zoonosis, and College of Veterinary Medicine, Jilin University, Changchun, 130062, China. Electronic address: zhanghaili@jlu.edu.cn.
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Harbin, 150069, China. Electronic address: wangxiaojun@caas.cn.
Conflict of Interest Statement
Declaration of Competing Interest The authors declare no conflicts of interest, financial or otherwise.
References
This article includes 35 references
- Baker S.F., Ledwith M.P., Mehle A. Differential splicing of anp32a in birds alters its ability to stimulate rna synthesis by restricted influenza polymerase. Cell Rep. 2018;24:2581–2588. e4.
- Bradel-Tretheway B.G., Mattiacio J.L., Krasnoselsky A., Stevenson C., Purdy D., Dewhurst S., Katze M.G. Comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and rna polymerase accessory factors. J. Virol. 2011;85:8569–8581.
- Brister H., Barnum S.M., Reedy S., Chambers T.M., Pusterla N. Validation of two multiplex real-time pcr assays based on single nucleotide polymorphisms of the ha1 gene of equine influenza a virus in order to differentiate between clade 1 and clade 2 Florida sublineage isolates. J. Vet. Diagn. Invest. 2019;31:137–141.
- Brody H. Influenza. Nature. 2019;573:S49.
- Carrique L., Fan H., Walker A.P., Keown J.R., Sharps J., Staller E., Barclay W.S., Fodor E., Grimes J.M. Host anp32a mediates the assembly of the influenza virus replicase. Nature. 2020;587:638–643.
- Chambers T.M. Equine influenza. Cold Spring Harb Perspect Med. 2022;12:a038331.
- Dao T.P., Majumdar A., Barrick D. Capping motifs stabilize the leucine-rich repeat protein pp32 and rigidify adjacent repeats. Protein Sci. 2014;23:801–811.
- Dao T.P., Majumdar A., Barrick D. Highly polarized c-terminal transition state of the leucine-rich repeat domain of pp32 is governed by local stability. Proc Natl Acad Sci U S A. 2015;112:E2298–E2306.
- Dilai M., Fassi Fihri O., El Harrak M., Bouchiba A., Dehhaoui M., Mahir W., Dikrallah A., Legrand L., Paillot R., Piro M. An evaluation of three different primary equine influenza vaccination intervals in foals. J. Equine Vet. Sci. 2021;99
- Domingues P., Hale B.G. Functional insights into anp32a-dependent influenza a virus polymerase host restriction. Cell Rep. 2017;20:2538–2546.
- Domingues P., Eletto D., Magnus C., Turkington H.L., Schmutz S., Zagordi O., Lenk M., Beer M., Stertz S., Hale B.G. Profiling host anp32a splicing landscapes to predict influenza a virus polymerase adaptation. Nat. Commun. 2019;10:3396.
- Fatima U., Zhang Z., Zhang H., Wang X.F., Xu L., Chu X., Ji S., Wang X. Equine mx1 restricts influenza a virus replication by targeting at distinct site of its nucleoprotein. Viruses. 2019;11:1114.
- Huang H. Roundup: equine influenza in U.S. kills 102 wild horses-xinhua. 2022. http://www.news.cn/world/2022-05/01/c_1128612971.htm Available from:
- Laabassi F., Lecouturier F., Amelot G., Gaudaire D., Mamache B., Laugier C., Legrand L., Zientara S., Hans A. Epidemiology and genetic characterization of h3n8 equine influenza virus responsible for clinical disease in Algeria in 2011. Transbound Emerg Dis. 2015;62:623–631.
- Lin Y., Wang X.F., Wang Y., Du C., Ren H., Liu C., Zhu D., Chen J., Na L., Liu D., Yang Z., Wang X. Env diversity-dependent protection of the attenuated equine infectious anaemia virus vaccine. Emerg. Microb. Infect. 2020;9:1309–1320.
- Long J.S., Giotis E.S., Moncorgé O., Frise R., Mistry B., James J., Morisson M., Iqbal M., Vignal A., Skinner M.A., Barclay W.S. Species difference in anp32a underlies influenza a virus polymerase host restriction. Nature. 2016;529:101–104.
- Long J.S., Idoko-Akoh A., Mistry B., Goldhill D., Staller E., Schreyer J., Ross C., Goodbourn S., Shelton H., Skinner M.A., Sang H., McGrew M.J., Barclay W. Species specific differences in use of anp32 proteins by influenza a virus. Elife. 2019;8
- Madić J., Martinović S., Naglić T., Hajsig D., Cvetnić S. Serological evidence for the presence of a/equine-1 influenza virus in unvaccinated horses in Croatia. Vet. Rec. 1996;138:68.
- Park Y.H., Woo S.J., Chungu K., Lee S.B., Shim J.H., Lee H.J., Kim I., Rengaraj D., Song C.S., Suh J.Y., Lim J.M., Han J.Y. Asp149 and asp152 in chicken and human anp32a play an essential role in the interaction with influenza viral polymerase. Faseb. J. 2021;35
- Peacock T.P., Swann O.C., Salvesen H.A., Staller E., Leung P.B., Goldhill D.H., Zhou H., Lillico S.G., Whitelaw C.B.A., Long J.S., Barclay W.S. Swine anp32a supports avian influenza virus polymerase. J. Virol. 2020;94:e00132–20.
- Rash A., Morton R., Woodward A., Maes O., McCauley J., Bryant N., Elton D. Evolution and divergence of h3n8 equine influenza viruses circulating in the United Kingdom from 2013 to 2015. Pathogens. 2017;6:6.
- Shapira S.D., Gat-Viks I., Shum B.O., Dricot A., de Grace M.M., Wu L., Gupta P.B., Hao T., Silver S.J., Root D.E., Hill D.E., Regev A., Hacohen N. A physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection. Cell. 2009;139:1255–1267.
- Spackman E. A brief introduction to the avian influenza virus. Methods Mol. Biol. 2008;436:1–6.
- Staller E., Sheppard C.M., Neasham P.J., Mistry B., Peacock T.P., Goldhill D.H., Long J.S., Barclay W.S. Anp32 proteins are essential for influenza virus replication in human cells. J. Virol. 2019;93:e00217–19.
- Sugiyama K., Kawaguchi A., Okuwaki M., Nagata K. Pp32 and april are host cell-derived regulators of influenza virus rna synthesis from crna. Elife. 2015;4
- Timoney P.J. Equine influenza. Comp. Immunol. Microbiol. Infect. Dis. 1996;19:205–211.
- van Maanen C., Cullinane A. Equine influenza virus infections: an update. Vet. Q. 2002;24:79–94.
- Wang F., Sheppard C.M., Mistry B., Staller E., Barclay W.S., Grimes J.M., Fodor E., Fan H. The c-terminal lcar of host anp32 proteins interacts with the influenza a virus nucleoprotein to promote the replication of the viral rna genome. Nucleic Acids Res. 2022;50:5713–5725.
- Wang J., Guo K., Li S., Liu D., Chu X., Wang Y., Guo W., Du C., Wang X., Hu Z. Development and application of real-time pcr assay for detection of salmonella abortusequi. J. Clin. Microbiol. 2023;61
- Webster R.G. Are equine 1 influenza viruses still present in horses? Equine Vet. J. 1993;25:537–538.
- Yamanaka T., Niwa H., Tsujimura K., Kondo T., Matsumura T. Epidemic of equine influenza among vaccinated racehorses in Japan in 2007. J. Vet. Med. Sci. 2008;70:623–625.
- Yu M., Qu Y., Zhang H., Wang X. Roles of anp32 proteins in cell biology and viral replication. Animal Diseases. 2022;2:22.
- Zhang H., Zhang Z., Wang Y., Wang M., Wang X., Zhang X., Ji S., Du C., Chen H., Wang X. Fundamental contribution and host range determination of anp32a and anp32b in influenza a virus polymerase activity. J. Virol. 2019;93:e00174–19.
- Zhang H., Li H., Wang W., Wang Y., Han G.Z., Chen H., Wang X. A unique feature of swine anp32a provides susceptibility to avian influenza virus infection in pigs. PLoS Pathog. 2020;16
- Zhang Z., Zhang H., Xu L., Guo X., Wang W., Ji Y., Lin C., Wang Y., Wang X. Selective usage of anp32 proteins by influenza b virus polymerase: implications in determination of host range. PLoS Pathog. 2020;16
Citations
This article has been cited 0 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists