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Preparation of Recombinant Alphaviruses for Functional Studies of ADP-Ribosylation

  • Rachy Abraham
  • Robert Lyle McPherson
  • Easwaran Sreekumar
  • Anthony K. L. Leung
  • Diane E. Griffin
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)

Abstract

Recently we characterized the mono(ADP-ribosyl) hydrolase (MAR hydrolase) activity of the macrodomain of nonstructural protein 3 (nsP3MD) of chikungunya virus. Using recombinant viruses with targeted mutations in the macrodomain, we demonstrated that hydrolase function is important for viral replication in cultured neuronal cells and for neurovirulence in mice. Here, we describe the general cell culture and animal model infection protocols for alphaviruses and the technical details for biochemical characterization of the MAR hydrolase activity of nsP3MD mutants and the preparation of recombinant viruses incorporating those mutations through site-directed mutagenesis of an infectious cDNA virus clone.

Key words

Alphavirus Chikungunya virus Sindbis virus MAR hydrolase assay Infectious cDNA clone Reverse genetics Site-directed mutagenesis In vitro transcription 

References

  1. 1.
    Schwartz O, Albert ML (2010) Biology and pathogenesis of chikungunya virus. Nat Rev Microbiol 8(7):491–500CrossRefGoogle Scholar
  2. 2.
    Burt FJ, Chen W, Miner JJ, Lenschow DJ, Merits A, Schnettler E, Kohl A, Rudd PA, Taylor A, Herrero LJ, Zaid A et al (2017) Chikungunya virus: an update on the biology and pathogenesis of this emerging pathogen. Lancet Infect Dis 17(4):e107–e117CrossRefGoogle Scholar
  3. 3.
    Strauss JH, Strauss EG (1994) The alphaviruses: gene expression, replication, and evolution. Microbiol Rev 58(3):491–562PubMedPubMedCentralGoogle Scholar
  4. 4.
    Rack JG, Perina D, Ahel I (2016) Macrodomains: structure, function, evolution, and catalytic activities. Annu Rev Biochem 85:431–454CrossRefGoogle Scholar
  5. 5.
    Gorbalenya AE, Koonin EV, Lai MM (1991) Putative papain-related thiol proteases of positive-strand RNA viruses. Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Lett 288(1–2):201–205CrossRefGoogle Scholar
  6. 6.
    Koonin EV, Gorbalenya AE, Purdy MA, Rozanov MN, Reyes GR, Bradley DW (1992) Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses. Proc Natl Acad Sci U S A 89(17):8259–8263CrossRefGoogle Scholar
  7. 7.
    McPherson RL, Abraham R, Sreekumar E, Ong SE, Cheng SJ, Baxter VK, Kistemaker HA, Filippov DV, Griffin DE, Leung AK (2017) ADP-ribosylhydrolase activity of Chikungunya virus macrodomain is critical for virus replication and virulence. Proc Natl Acad Sci 114(7):1666–1671CrossRefGoogle Scholar
  8. 8.
    Kümmerer BM, Grywna K, Gläsker S, Wieseler J, Drosten C (2012) Construction of an infectious Chikungunya virus cDNA clone and stable insertion of mCherry reporter genes at two different sites. J Gen Virol 93(9):1991–1995CrossRefGoogle Scholar
  9. 9.
    Scholte FE, Tas A, Martina BE, Cordioli P, Narayanan K, Makino S, Snijder EJ, van Hemert MJ (2013) Characterization of synthetic Chikungunya viruses based on the consensus sequence of recent E1-226V isolates. PLoS One 8(8):e71047CrossRefGoogle Scholar
  10. 10.
    Tsetsarkin K, Higgs S, McGee CE, Lamballerie XD, Charrel RN, Vanlandingham DL (2006) Infectious clones of Chikungunya virus (La Reunion isolate) for vector competence studies. Vector-Borne Zoonotic Dis 6(4):325–337CrossRefGoogle Scholar
  11. 11.
    Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S (2007) A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 3(12):e201CrossRefGoogle Scholar
  12. 12.
    Tsetsarkin KA, McGee CE, Volk SM, Vanlandingham DL, Weaver SC, Higgs S (2009) Epistatic roles of E2 glycoprotein mutations in adaption of chikungunya virus to Aedes albopictus and Ae. aegypti mosquitoes. PLoS One 4(8):e6835CrossRefGoogle Scholar
  13. 13.
    Vanlandingham DL, Tsetsarkin K, Hong C, Klingler K, McElroy KL, Lehane MJ, Higgs S (2005) Development and characterization of a double subgenomic chikungunya virus infectious clone to express heterologous genes in Aedes aegypti mosqutioes. Insect Biochem Mol Biol 35(10):1162–1170CrossRefGoogle Scholar
  14. 14.
    Plante K, Wang E, Partidos CD, Weger J, Gorchakov R, Tsetsarkin K, Borland EM, Powers AM, Seymour R, Stinchcomb DT, Osorio JE (2011) Novel chikungunya vaccine candidate with an IRES-based attenuation and host range alteration mechanism. PLoS Pathog 7(7):e1002142CrossRefGoogle Scholar
  15. 15.
    Gorchakov R, Wang E, Leal G, Forrester NL, Plante K, Rossi SL, Partidos CD, Adams AP, Seymour RL, Weger J, Borland EM (2012) Attenuation of Chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the E2 envelope glycoprotein. J Virol 86(11):6084–6096CrossRefGoogle Scholar
  16. 16.
    Wang E, Weaver SC, Frolov I (2011) Chimeric Chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response. J Virol 85(17):9249–9252CrossRefGoogle Scholar
  17. 17.
    Tretyakova I, Hearn J, Wang E, Weaver S, Pushko P (2014) DNA vaccine initiates replication of live attenuated chikungunya virus in vitro and elicits protective immune response in mice. J Infect Dis 209:1882–1890CrossRefGoogle Scholar
  18. 18.
    Levitt NH, Ramsburg HH, Hasty SE, Repik PM, Cole FE, Lupton HW (1986) Development of an attenuated strain of chikungunya virus for use in vaccine production. Vaccine 4(3):157–162CrossRefGoogle Scholar
  19. 19.
    Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, Shaw IT, Dahrouge S, Antel JP (1992) Neuroblastoma× spinal cord (NSC) hybrid cell lines resemble developing motor neurons. Dev Dyn 194(3):209–221CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Rachy Abraham
    • 1
  • Robert Lyle McPherson
    • 2
  • Easwaran Sreekumar
    • 1
    • 3
  • Anthony K. L. Leung
    • 2
    • 4
    • 5
  • Diane E. Griffin
    • 1
  1. 1.Department of Molecular Microbiology and ImmunologyBloomberg School of Public Health, Johns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Biochemistry and Molecular BiologyBloomberg School of Public Health, Johns Hopkins UniversityBaltimoreUSA
  3. 3.Viral Disease Biology ProgramRajiv Gandhi Centre for Biotechnology (RGCB)ThiruvananthapuramIndia
  4. 4.Department of OncologySchool of Medicine, Johns Hopkins UniversityBaltimoreUSA
  5. 5.Department of Molecular Biology and GeneticsSchool of Medicine, Johns Hopkins UniversityBaltimoreUSA

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