FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Viruses / Production of Recombinant EAV with Tagged Structural Protein...
Viruses2019; 11(8); doi: 10.3390/v11080735

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Abstract: Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.
Get Full Text
Publication Date: 2019-08-09 PubMed ID: 31404947PubMed Central: PMC6723265DOI: 10.3390/v11080735Google 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.
LinkedInCopy
  • Journal Article
  • Research Support
  • Non-U.S. Gov't

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.

The study involved the creation of a variant of the Equine arteritis virus (EAV) with a modified glycoprotein, Gp3, to analyze its role in virus entry and cell infection. The research suggests that this altered virus could serve as a valuable tool for investigating host responses to infection and the function of specific proteins during viral attachment and entry.

Understanding the Objectives of the Study

  • The core goal of this research was to generate a derivative of the Equine arteritis virus (EAV) having separated open reading frames 3 and 4, and a Gp3 protein with the HA-tag.
  • Another objective of the study was to use this modified EAV in examining where the Gp3-HA, an important minor protein of Arterivirus, is found in infected cells.
  • The modified EAV may facilitate improved understanding of how the Gp3 protein influences the virus’s attachment and entry into cells, as well as how the host cell’s factors interact during infection.

Study Methodology

  • Researchers generated a version of EAV virus that incorporated alterations to its genetic composition, specifically its open reading frames 3 and 4, and its Gp3 protein.
  • The modified protein, Gp3-HA, was designed to be recognized by antibodies, enabling researchers to track its location in infected cells.
  • Scientists monitored the stability of the virus over multiple passages and its replicative capacity in comparison to the wild-type EAV.

Main Findings of the Research

  • The modified virus remained stable during passaging and demonstrated a similar capability to multiply as the non-modified version.
  • The research revealed that Gp3-HA is integrated into the viral particles both as independent proteins and as part of a three-protein complex, Gp2/Gp3-HA/Gp4.
  • Localization studies showed that Gp3-HA is found within the endoplasmic reticulum and, to a lesser degree, within the Golgi apparatus of infected cells. It was also discovered that Gp3-HA co-localizes with the E protein, but not the N protein.
  • The researchers also found that the co-localization of Gp3-HA and the E protein with the certain organelles was reduced.

Implications of the Study

  • This research provides invaluable insights into the role and behavior of Arterivirus minor proteins during an infection.
  • The findings offer a potential path towards the development of better diagnostic tools or treatments by shedding light on the interaction between host cell factors and the virus during infection.
  • Further research armed with this modified EAV would allow a more detailed understanding of how the Gp3 protein influences virus attachment and entry, which could prove crucial in combating diseases caused by Arteriviruses.

Cite This Article

APA
Matczuk AK, Chodaczek G, Ugorski M. (2019). Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells. Viruses, 11(8). https://doi.org/10.3390/v11080735

Publication

Viruses
ISSN: 1999-4915
NlmUniqueID: 101509722
Country: Switzerland
Language: English
Volume: 11
Issue: 8

Researcher Affiliations

Matczuk, Anna Karolina
  • Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław 50-375, Poland. anna.matczuk@upwr.edu.pl.
  • Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław 50-375, Poland. anna.matczuk@upwr.edu.pl.
Chodaczek, Grzegorz
  • Confocal Microscopy Laboratory, PORT Polish Center for Technology Development, Wrocław 54-066, Poland.
Ugorski, Maciej
  • Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław 50-375, Poland.

MeSH Terms

  • Animals
  • Arterivirus Infections / veterinary
  • Cell Line
  • Equartevirus / genetics
  • Equartevirus / metabolism
  • Genetic Engineering
  • Genome, Viral
  • Golgi Apparatus / metabolism
  • Horse Diseases / virology
  • Horses
  • Host-Pathogen Interactions
  • Intracellular Space
  • Mutation
  • Open Reading Frames
  • Protein Transport
  • Viral Envelope Proteins / genetics
  • Viral Envelope Proteins / metabolism
  • Virus Replication

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 38 references
  1. Snijder EJ, Kikkert M, Fang Y. Arterivirus molecular biology and pathogenesis.. J. Gen. Virol. 2013;94:2141–2163.
    doi: 10.1099/vir.0.056341-0pubmed: 23939974google scholar: lookup
  2. MacLachlan NJ, Balasuriya UB. Equine Viral Arteritis.. In: Perlman S., Holmes K.V., editors. Proceedings of the The Nidoviruses. Springer US; Boston, MA, USA: 2006. pp. 429–433.
    pmc: PMC7123899pubmed: 17037573
  3. Den Boon JA, Snijder EJ, Chirnside ED, de Vries AA, Horzinek MC, Spaan WJ. Equine arteritis virus is not a togavirus but belongs to the coronaviruslike superfamily.. J. Virol. 1991;65:2910–2920.
    pmc: PMC240924pubmed: 1851863
  4. De Vries AA, Chirnside ED, Bredenbeek PJ, Gravestein LA, Horzinek MC, Spaan WJ. All subgenomic mRNAs of equine arteritis virus contain a common leader sequence.. Nucleic Acids Res. 1990;18:3241–3247.
    doi: 10.1093/nar/18.11.3241pmc: PMC330929pubmed: 2162519google scholar: lookup
  5. Veit M, Matczuk AK, Sinhadri BC, Krause E, Thaa B. Membrane proteins of arterivirus particles: structure, topology, processing and function.. Virus Res. 2014;194:16–36.
    doi: 10.1016/j.virusres.2014.09.010pmc: PMC7172906pubmed: 25278143google scholar: lookup
  6. Wieringa R, de Vries AAF, van der Meulen J, Godeke G-J, Onderwater JJM, van Tol H, Koerten HK, Mommaas AM, Snijder EJ, Rottier PJM. Structural Protein Requirements in Equine Arteritis Virus Assembly.. J. Virol. 2004;78:13019–13027.
    doi: 10.1128/JVI.78.23.13019-13027.2004pmc: PMC524988pubmed: 15542653google scholar: lookup
  7. Guo J, Tian J, Tan X, Yu H, Ding S, Sun H, Yu X. Pathological observations of an experimental infection of geese with goose circovirus.. Avian Pathol. 2011;40:55–61.
    doi: 10.1080/03079457.2010.538371pubmed: 21331948google scholar: lookup
  8. Das PB, Dinh PX, Ansari IH, de Lima M, Osorio FA, Pattnaik AK. The Minor Envelope Glycoproteins GP2a and GP4 of Porcine Reproductive and Respiratory Syndrome Virus Interact with the Receptor CD163.. J. Virol. 2010;84:1731–1740.
    doi: 10.1128/JVI.01774-09pmc: PMC2812361pubmed: 19939927google scholar: lookup
  9. Burkard C, Opriessnig T, Mileham AJ, Stadejek T, Ait-Ali T, Lillico SG, Whitelaw CBA, Archibald AL. Pigs Lacking the Scavenger Receptor Cysteine-Rich Domain 5 of CD163 Are Resistant to Porcine Reproductive and Respiratory Syndrome Virus 1 Infection.. J. Virol. 2018;92:e00415-18.
    doi: 10.1128/JVI.00415-18pmc: PMC6069206pubmed: 29925651google scholar: lookup
  10. Lee C, Yoo D. The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties.. Virology. 2006;355:30–43.
    doi: 10.1016/j.virol.2006.07.013pmc: PMC7111972pubmed: 16904148google scholar: lookup
  11. Wieringa R, de Vries AAF, Rottier PJM. Formation of Disulfide-Linked Complexes between the Three Minor Envelope Glycoproteins of Equine Arteritis Virus.. J. Virol. 2003;77:6216–6226.
    doi: 10.1128/JVI.77.11.6216-6226.2003pmc: PMC155002pubmed: 12743278google scholar: lookup
  12. Snijder EJ, van Tol H, Pedersen KW, Raamsman MJB, de Vries AAF. Identification of a Novel Structural Protein of Arteriviruses.. J. Virol. 1999;73:6335–6345.
    pmc: PMC112712pubmed: 10400725
  13. Wieringa R, de Vries AAF, Raamsman MJB, Rottier PJM. Characterization of Two New Structural Glycoproteins, GP3 and GP4, of Equine Arteritis Virus.. J. Virol. 2002;76:10829–10840.
    doi: 10.1128/JVI.76.21.10829-10840.2002pmc: PMC136612pubmed: 12368326google scholar: lookup
  14. Magnusson P, Hyllseth B, Marusyk H. Morphological studies on equine arteritis virus.. Arch. Gesamte Virusforsch. 1970;30:105–112.
    doi: 10.1007/BF01250177pubmed: 4195609google scholar: lookup
  15. Wood O, Tauraso N, Liebhaber H. Electron microscopicstudy of tissue cultures infected with simian haemorrhagic fever virus.. J. Gen. Virol. 1970;7:129–136.
    doi: 10.1099/0022-1317-7-2-129pubmed: 4987738google scholar: lookup
  16. Snijder EJ, van Tol H, Roos N, Pedersen KW. Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex.. J. Gen. Virol. 2001;82:985–994.
    doi: 10.1099/0022-1317-82-5-985pubmed: 11297673google scholar: lookup
  17. Van den Born E, Posthuma CC, Gultyaev AP, Snijder EJ. Discontinuous Subgenomic RNA Synthesis in Arteriviruses Is Guided by an RNA Hairpin Structure Located in the Genomic Leader Region.. J. Virol. 2005;79:6312–6324.
    doi: 10.1128/JVI.79.10.6312-6324.2005pmc: PMC1091703pubmed: 15858015google scholar: lookup
  18. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B. Fiji: An open-source platform for biological-image analysis.. Nat. Methods. 2012;9:676.
    doi: 10.1038/nmeth.2019pmc: PMC3855844pubmed: 22743772google scholar: lookup
  19. Bolte S, Cordelieres FP. A guided tour into subcellular colocalization analysis in light microscopy.. J. Microsc. 2006;224:213–232.
    doi: 10.1111/j.1365-2818.2006.01706.xpubmed: 17210054google scholar: lookup
  20. Matczuk AK, Kunec D, Veit M. Co-translational Processing of Glycoprotein 3 from Equine Arteritis Virus: N-Glycosylation Adjacent To The Signal Peptide Prevents Cleavage.. J. Biol. Chem. 2013;288:35396–35405.
    doi: 10.1074/jbc.M113.505420pmc: PMC3853287pubmed: 24142700google scholar: lookup
  21. Matczuk AK, Ugorski M. Expression of recombinant, tagged GP2/3/4 trimer and E protein from equine arteritis virus in eukaryotic system; Proceedings of the 28th Annual Meeting of the Society for Virology; Wurzburg, Germany.. 14–17 March 2018.
  22. Matczuk AK, Veit M. Signal peptide cleavage from GP3 enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic C-terminus is essential.. Virus Res. 2014;183:107–111.
    doi: 10.1016/j.virusres.2014.02.005pubmed: 24556360google scholar: lookup
  23. De Vries AAF, Glaser AL, Raamsman MJB, de Haan CAM, Sarnataro S, Godeke G-J, Rottier PJM. Genetic Manipulation of Equine Arteritis Virus Using Full-Length cDNA Clones: Separation of Overlapping Genes and Expression of a Foreign Epitope.. Virology. 2000;270:84–97.
    doi: 10.1006/viro.2000.0245pubmed: 10772982google scholar: lookup
  24. Van den Born E, Posthuma CC, Knoops K, Snijder EJ. An infectious recombinant equine arteritis virus expressing green fluorescent protein from its replicase gene.. J. Gen. Virol. 2007;88:1196–1205.
    doi: 10.1099/vir.0.82590-0pubmed: 17374763google scholar: lookup
  25. Mondal SP, Cook RF, Chelvarajan RL, Henney PJ, Timoney PJ, Balasuriya UBR. Development and characterization of a synthetic infectious cDNA clone of the virulent Bucyrus strain of equine arteritis virus expressing mCherry (red fluorescent protein). Arch. Virol. 2016;161:821–832.
    doi: 10.1007/s00705-015-2633-6pubmed: 26711457google scholar: lookup
  26. Lum KK, Cristea IM. Proteomic approaches to uncovering virus–host protein interactions during the progression of viral infection.. Expert Rev. Proteomics. 2016;13:325–340.
    doi: 10.1586/14789450.2016.1147353pmc: PMC4919574pubmed: 26817613google scholar: lookup
  27. Renukaradhya GJ, Meng X-J, Calvert JG, Roof M, Lager KM. Live porcine reproductive and respiratory syndrome virus vaccines: Current status and future direction.. Vaccine. 2015;33:4069–4080.
    doi: 10.1016/j.vaccine.2015.06.092pubmed: 26148878google scholar: lookup
  28. Tijms MA, van der Meer Y, Snijder EJ. Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus.. J. Gen. Virol. 2002;83:795–800.
    doi: 10.1099/0022-1317-83-4-795pubmed: 11907328google scholar: lookup
  29. Ellgaard L, Helenius A. Quality control in the endoplasmic reticulum.. Nat. Rev. Mol. Cell Biol. 2003;4:181.
    doi: 10.1038/nrm1052pubmed: 12612637google scholar: lookup
  30. De Vries AA, Post SM, Raamsman MJ, Horzinek MC, Rottier PJ. The two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers.. J. Virol. 1995;69:4668–4674.
    pmc: PMC189270pubmed: 7609031
  31. Vennema H, Godeke GJ, Rossen JW, Voorhout WF, Horzinek MC, Opstelten DJ, Rottier PJ. Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes.. EMBO J. 1996;15:2020–2028.
    doi: 10.1002/j.1460-2075.1996.tb00553.xpmc: PMC450121pubmed: 8617249google scholar: lookup
  32. Ujike M, Taguchi F. Incorporation of Spike and Membrane Glycoproteins into Coronavirus Virions.. Viruses. 2015;7:1700–1725.
    doi: 10.3390/v7041700pmc: PMC4411675pubmed: 25855243google scholar: lookup
  33. Klumperman J, Locker JK, Meijer A, Horzinek MC, Geuze HJ, Rottier PJ. Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.. J. Virol. 1994;68:6523–6534.
    pmc: PMC237073pubmed: 8083990
  34. Nal B, Chan C, Kien F, Siu L, Tse J, Chu K, Kam J, Staropoli I, Crescenzo-Chaigne B, Escriou N. Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.. J. Gen. Virol. 2005;86:1423–1434.
    doi: 10.1099/vir.0.80671-0pubmed: 15831954google scholar: lookup
  35. Dobbe JC, van der Meer Y, Spaan WJM, Snijder EJ. Construction of Chimeric Arteriviruses Reveals That the Ectodomain of the Major Glycoprotein Is Not the Main Determinant of Equine Arteritis Virus Tropism in Cell Culture.. Virology. 2001;288:283–294.
    doi: 10.1006/viro.2001.1074pubmed: 11601900google scholar: lookup
  36. Venkatagopalan P, Daskalova SM, Lopez LA, Dolezal KA, Hogue BG. Coronavirus envelope (E) protein remains at the site of assembly.. Virology. 2015;478:75–85.
    doi: 10.1016/j.virol.2015.02.005pmc: PMC4550588pubmed: 25726972google scholar: lookup
  37. Veit M, Thaa B. Association of influenza virus proteins with membrane rafts.. Adv. Virol. 2011;2011:370606.
    doi: 10.1155/2011/370606pmc: PMC3265303pubmed: 22312341google scholar: lookup
  38. Leser GP, Lamb RA. Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.. J. Virol. 2017;91:e02104-16.
    doi: 10.1128/JVI.02104-16pmc: PMC5391459pubmed: 28202765google scholar: lookup

Citations

This article has been cited 1 times.
  1. Matczuk AK, Zhang M, Veit M, Ugorski M. Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells. Viruses 2022 Apr 1;14(4).
    doi: 10.3390/v14040749pubmed: 35458479google scholar: lookup
Subscribe to our newsletter
Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.