Abstract
Cytoplasmic replication of poxviruses dictates the encoding of most, if not all, of the trans-acting factors required for faithful genome duplication. Several of these proteins have been identified through genetic and biochemical evaluation, including the catalytic DNA polymerase (E9), an essential and stoichiometric component of the processive polymerase (A20), a singlestrand DNA-binding protein (I3), a type I topoisomerase (H6), the uracil DNA glycosylase (D4), a nucleic acid-independent nucleoside triphosphatase (D5), a serine/threonine protein kinase (B1), and a Holliday Junction resolvase (A22). All of these factors work in concert to faithfully duplicate the viral genome. Although a replication origin has not been defined for the poxviruses, cis-acting sequences found within the telomeric 200 bp have been implicated as necessary and sufficient for minichromosome replication. Replication occurs within cytoplasmic foci from approx 3 to 12 h postinfection. This chapter includes several methodologies to assay and quantitate replication in vivo, visualize replication foci microscopically, and test the integrity of central replication enzymes in vitro.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Moss, B. (2001) Poxviridae: the viruses and their replication, in Fields Virology (Knipe, D. M. and Howley, P. M., eds.), Lippincott-Raven, P□A, pp. 2849–2884.
Traktman, P. (1996) Poxvirus DNA replication, in DNA Replication in Eukaryotic Cells (Depamphilis, M. L., ed.), Cold Spring Harbor Laboratory, Cold Spring, NY, pp. 775–798.
Du, S._ and Traktman, P. (1996) Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates. Proc. Natl. Acad. Sci. USA 93, 9693–9698.
Evans, E. and Traktman, P. (1992) Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene. Chromosoma 102, S72–S82.
Punjabi, A., Boyle, K., DeMasi, J., Grubisha, O., Unger, B., Khanna, M., and Traktman, P. (2001) Clustered charge-to-alanine mutagenesis of the vaccinia virus A20 gene: temperature-sensitive mutants have a DNA-minus phenotype and are defective in the production of processive DNA polymerase activity. J. Virol. 75, 12308–12318.
Rempel, R. E., Anderson, M. K., Evans, E., and Traktman, P. (1990) Temperature-sensitive vaccinia virus mutants identify a gene with an essential role in viral replication. J. Virol. 64, 574–583.
Brown, T. (1994) Enzymatic manipulation of DNA and RNA, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds.), John Wiley & Sons, Inc., Cambridge, MA, pp. 2.9.1–2.9.15.
Domi, A. and Beaud, G. (2000) The punctate sites of accumulation of vaccinia virus early proteins are precursors of sites of viral DNA synthesis. J. Gen. Virol. 81, 1231–1235.
Garcia, A. D. and Moss, B. (2001) Repression of vaccinia virus Holliday junction resolvase inhibits processing of viral DNA into unit-length genomes. J. Virol. 75, 6460–6471.
Merchlinsky, M. and Moss, B. (1989) Resolution of vaccinia virus DNA concatemer junctions requires late-gene expression. J. Virol. 63, 1595–1603.
Merchlinsky, M. and Moss, B. (1986) Resolution of linear minichromosomes with hairpin ends from circular plasmids containing vaccinia virus concatemer junctions. Cell 45, 879–884.
Tabor, S. and Struhl, K. (1994) Preparation and analysis of DNA, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds.), John Wiley & Sons, Inc., Cambridge, MA, pp. 3.5.4–3.5.10.
DeLange, A. M. (1989) Identification of temperature-sensitive mutants of vaccinia virus that are defective in conversion of concatemeric replicative intermediates to the mature linear DNA genome. J. Virol. 63, 2437–2444.
Baroudy, B. M., Venkatesan, S., and Moss, B. (1982) Incompletely base-paired flip-flop terminal loops link the two DNA strands of the vaccinia virus genome into one uninterrupted polynucleotide chain. Cell 28, 315–324.
DeMasi, J., Du, S., Lennon, D., and Traktman, P. (2001) Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins. J. Virol. 75, 10090–10105.
Klemperer, N., McDonald, W., Boyle, K., Unger, B., and Traktman, P. (2001) The A20R protein is a stoichiometric component of the processive form of vaccinia virus DNA polymerase. J. Virol. 75, 12298–12307.
McDonald, W. F. and Traktman, P. (1994) Vaccinia virus DNA polymerase. In vitro analysis of parameters affecting processivity. J. Biol. Chem. 269, 31190–31197.
McDonald, W. F., Klemperer, N., and Traktman, P. (1997) Characterization of a processive form of the vaccinia virus DNA polymerase. Virology 234, 168–175.
McDonald, W. F. and Traktman, P. (1994) Overexpression and purification of the vaccinia virus DNA polymerase. Protein Expr. Purif. 5, 409–421.
Zhang, Y., Keck, J. G., and Moss, B. (1992) Transcription of viral late genes is dependent on expression of the viral intermediate gene G8R in cells infected with an inducible conditional-lethal mutant vaccinia virus. J. Virol. 66, 6470–6479.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Traktman, P., Boyle, K. (2004). Methods for Analysis of Poxvirus DNA Replication. In: Isaacs, S.N. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 269. Humana Press. https://doi.org/10.1385/1-59259-789-0:169
Download citation
DOI: https://doi.org/10.1385/1-59259-789-0:169
Publisher Name: Humana Press
Print ISBN: 978-1-58829-229-2
Online ISBN: 978-1-59259-789-5
eBook Packages: Springer Protocols