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Rhinoviruses pp 171-180 | Cite as

Reverse Genetic Engineering of the Human Rhinovirus Serotype 16 Genome to Introduce an Antibody-Detectable Tag

  • Erin J. WalkerEmail author
  • Lora M. Jensen
  • Reena Ghildyal
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1221)

Abstract

The ability to accurately detect viral proteins during infection is essential for virology research, and the lack of specific antibodies can make this detection difficult. Reverse genetic engineering of virus genomes to alter the wild-type genome is a powerful technique to introduce a detectable tag onto a viral protein. Here we outline a method to incorporate an influenza hemagglutinin epitope tag onto the 2A protease of HRV16. The method uses site-directed mutagenesis PCR to introduce the sequence for the HA antigen onto either the C or N termini of 2A protease while keeping the relevant internal cleavage sites intact. The new viral product is then cloned into a wild-type HRV16 plasmid and transfected into Ohio Hela cells to produce recombinant virus.

Key words

Human rhinovirus Reverse genetic engineering 

Notes

Acknowledgements

We thank Dr Wai-Ming Lee for the kind gift of anti-VP2 antibodies and the pR16.11 HRV16 plasmid.

References

  1. 1.
    Johnston SL et al (1995) Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. BMJ 310(6989):1225–1229PubMedCrossRefPubMedCentralGoogle Scholar
  2. 2.
    Kaur B et al (1998) Prevalence of asthma symptoms, diagnosis, and treatment in 12–14 year old children across Great Britain (international study of asthma and allergies in childhood, ISAAC UK). BMJ 316(7125):118–124PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Younessi P, Jans DA, Ghildyal R (2012) Modulation of host cell nucleocytoplasmic trafficking during picornavirus infection. Infect Disord Drug Targets 12(1):59–67PubMedCrossRefGoogle Scholar
  4. 4.
    Racaniello VR (2001) Picornaviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields virology. Lippincott/Raven, Philadelphia, PA, pp 685–722Google Scholar
  5. 5.
    Lee WM, Wang W (2003) Human rhinovirus type 16: mutant V1210A requires capsid-binding drug for assembly of pentamers to form virions during morphogenesis. J Virol 77(11):6235–6244PubMedCrossRefPubMedCentralGoogle Scholar
  6. 6.
    Reed LJ, Muench H (1938) A simple method of estimation fifty percent endpoints. Am J Hyg 27(5):493–497Google Scholar
  7. 7.
    Walker EJ et al (2013) Rhinovirus 3C protease facilitates specific nucleoporin cleavage and mislocalisation of nuclear proteins in infected host cells. PLoS One 8(8):e71316PubMedCrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Erin J. Walker
    • 1
    Email author
  • Lora M. Jensen
    • 1
  • Reena Ghildyal
    • 1
  1. 1.Centre for Research in Therapeutic Solutions, Faculty of Education, Science, Technology and MathematicsUniversity of CanberraCanberraAustralia

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