Skip to main content
SpringerLink
Log in

Generation of Vaccinia Virus Gene Deletion Mutants Using Complementing Cell Lines

  • Protocol
  • First Online:
Vaccinia Virus

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2023))

Abstract

This protocol describes how to couple two techniques, the generation of complementing cells lines and production of viral deletion mutants, to rapidly construct novel tools for poxvirus analysis. Specifically, the production and utilization of a complementing cell line expressing a poxvirus gene of interest are critical for the generation of poxvirus mutants in which essential genes are disrupted. Complementing cells are also valuable for the characterization of vaccinia genes in the absence of infection. Here, we detail the process of isolating vaccinia virus deletion mutants. Deletion mutant generation involves homologous recombination between replicating viral DNA and transfected DNA followed by selection and screening on a complementing cell line that provides the deleted gene in trans. Finally, deletion is confirmed by polymerase chain reaction, sequencing, and functional assays if available.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mackett M, Smith GL, Moss B (1982) Vaccinia virus: a selectable eukaryotic cloning and expression vector. Proc Natl Acad Sci 79(23):7415–7419

    Article  CAS  Google Scholar 

  2. Blasco R, Moss B (1991) Extracellular vaccinia virus formation and cell-to-cell virus transmission are prevented by deletion of the gene encoding the 37,000-Dalton outer envelope protein. J Virol 65(11):5910–5920

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Blasco R, Cole NB, Moss B (1991) Sequence analysis, expression, and deletion of a vaccinia virus gene encoding a homolog of profilin, a eukaryotic actin-binding protein. J Virol 65(9):4598–4608

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Mackett M, Smith GL, Moss B (1984) General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49(3):857–864

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Roper RL, Wolffe EJ, Weisberg A et al (1998) The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell-to-cell spread of vaccinia virus. J Virol 72(5):4192–4204

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Chernos VI, Belanov EF, Vasilieva NN (1978) Temperature-sensitive mutants of vaccinia virus. I. Isolation and preliminary characterization. Acta Virol 22(2):81–90

    CAS  PubMed  Google Scholar 

  7. Condit RCMA (1981) Isolation and preliminary characterization of temperature-sensitive mutants of vaccinia virus. Virology 113(1):224–241

    Article  CAS  Google Scholar 

  8. Rempel RE, Anderson MK, Evans E et al (1990) Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication. J Virol 64(2):574–583

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Evans ETP (1992) Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene. Chromosoma 102(1):S72–S82

    Article  CAS  Google Scholar 

  10. da Fonseca FG, Wolffe EJ, Weisberg A, Moss B (2000) Effects of deletion or stringent repression of the H3L envelope gene on vaccinia virus replication. J Virol 74(16):7518–7528

    Article  Google Scholar 

  11. Szajner P, Weisberg AS, Moss B (2004) Evidence for an essential catalytic role of the F10 protein kinase in vaccinia virus morphogenesis. J Virol 78(1):257–265

    Article  CAS  Google Scholar 

  12. Meng X, Wu X, Yan B, Deng J, Xiang Y (2013) Analysis of the role of vaccinia virus H7 in virion membrane biogenesis with an H7-deletion mutant. J Virol 87(14):8247–8253. https://doi.org/10.1128/JVI.00845-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Maruri-Avidal L, Weisberg AS, Bisht H, Moss B (2013) Analysis of viral membranes formed in cells infected by a vaccinia virus L2-deletion mutant suggests their origin from the endoplasmic reticulum. J Virol 87(3):1861–1871. https://doi.org/10.1128/JVI.02779-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Boyle KA, Greseth MD, Traktman P (2015) Genetic confirmation that the H5 protein is required for vaccinia virus DNA replication. J Virol 89:6312–6327

    Article  CAS  Google Scholar 

  15. Warren RD, Cotter CA, Moss B (2012) Reverse genetics analysis of poxvirus intermediate transcription factors. J Virol 86(17):9514–9519. https://doi.org/10.1128/JVI.06902-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kolli S, Meng X, Wu X, Shengjuler D, Cameron CE, Xiang Y (2015) Structure-function analysis of vaccinia virus H7 protein reveals a novel phosphoinositide binding fold essential for poxvirus replication. J Virol 89(4):2209–2219. https://doi.org/10.1128/JVI.03073-14

    Article  CAS  PubMed  Google Scholar 

  17. Hyun SI, Weisberg A, Moss B (2017) Deletion of the vaccinia virus I2 protein interrupts virion morphogenesis, leading to retention of the scaffold protein and mislocalization of membrane-associated entry proteins. J Virol 91(15):e00558–e00517. https://doi.org/10.1128/JVI.00558-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Meng X, Rose L, Han Y, Deng J, Xiang Y (2017) Vaccinia virus A6 is a two-domain protein requiring a cognate N-terminal domain for full viral membrane assembly activity. J Virol 91(10):e02405–e02416. https://doi.org/10.1128/JVI.02405-16

    Article  PubMed  PubMed Central  Google Scholar 

  19. Maruri-Avidal L, Weisberg AS, Moss B (2013) Direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized L2-interacting protein A30.5. J Virol 87(22):12313–12326. https://doi.org/10.1128/JVI.02137-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sutter G, Ramsey-Ewing A, Rosales R, Moss B (1994) Stable expression of the vaccinia virus K1L gene in rabbit cells complements the host range defect of a vaccinia virus mutant. J Virol 68(7):4109–4116

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Olson AT, Rico AB, Wang Z, Delhon G, Wiebe MS (2017) Deletion of the vaccinia virus B1 kinase reveals essential functions of this enzyme complemented partly by the homologous cellular kinase VRK2. J Virol 91(15):e00635–e00617. https://doi.org/10.1128/JVI.00635-17

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Borrego B, Lorenzo MM, Blasco R (1999) Complementation of P37 (F13L gene) knock-out in vaccinia virus by a cell line expressing the gene constitutively. J Gen Virol 80(Pt. 2):425–432. https://doi.org/10.1099/0022-1317-80-2-425

    Article  CAS  PubMed  Google Scholar 

  23. Holzer GW, Falkner FG (1997) Construction of a vaccinia virus deficient in the essential DNA repair enzyme uracil DNA glycosylase by a complementing cell line. J Virol 71(7):4997–5002

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Schambach A, Zychlinski D, Ehrnstroem B, Baum C (2013) Biosafety features of lentiviral vectors. Hum Gene Ther 24(2):132–142. https://doi.org/10.1089/hum.2012.229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chakrabarti S, Sisler JR, Moss B (1997) Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23(6):1094–1097

    Article  CAS  Google Scholar 

  26. Davison AJ, Moss B (1990) New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. Nucleic Acids Res 18(14):4285–4286

    Article  CAS  Google Scholar 

  27. Bertholet C, Drillien R, Wittek R (1985) One hundred base pairs of 5′ flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription. Proc Natl Acad Sci U S A 82(7):2096–2100

    Article  CAS  Google Scholar 

  28. Wittek R, Hanggi M, Hiller G (1984) Mapping of a gene coding for a major late structural polypeptide on the vaccinia virus genome. J Virol 49(2):371–378

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Cotter CA, Earl PL, Wyatt LS, Moss B (2017) Preparation of cell cultures and vaccinia virus stocks. Curr Protoc Protein Sci 89:5.12.1–5.12.18. https://doi.org/10.1002/cpps.34

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. School of Veterinary Medicine and Biomedical Sciences, University of Nebraska, Lincoln, NE, USA

    Amber B. Rico & Matthew S. Wiebe

  2. Nebraska Center for Virology, University of Nebraska, Lincoln, NE, USA

    Amber B. Rico, Annabel T. Olson & Matthew S. Wiebe

  3. School of Biological Sciences, University of Nebraska, Lincoln, NE, USA

    Annabel T. Olson

Authors
  1. Amber B. Rico
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Annabel T. Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Matthew S. Wiebe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthew S. Wiebe .

Editor information

Editors and Affiliations

  1. MRC-Laboratory for Molecular Cell Biology, University College London, London, UK

    Jason Mercer

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Rico, A.B., Olson, A.T., Wiebe, M.S. (2019). Generation of Vaccinia Virus Gene Deletion Mutants Using Complementing Cell Lines. In: Mercer, J. (eds) Vaccinia Virus. Methods in Molecular Biology, vol 2023. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9593-6_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9593-6_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9592-9

  • Online ISBN: 978-1-4939-9593-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation