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

Punjabi, A., Boyle, K., DeMasi, J., Grubisha, O. ORCID: https://orcid.org/0000-0003-1067-1230, 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. Journal of Virology 75 (24) , pp. 12308-12318. 10.1128/JVI.75.24.12308-12318.2001

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Abstract

Although the vaccinia virus DNA polymerase is inherently distributive, a highly processive form of the enzyme exists within the cytoplasm of infected cells (W. F. McDonald, N. Klemperer, and P. Traktman, Virology 234:168–175, 1997). In the accompanying report we outline the purification of the 49-kDa A20 protein as a stoichiometric component of the processive polymerase complex (N. Klemperer, W. McDonald, K. Boyle, B. Unger, and P. Traktman, J. Virol. 75:12298–12307, 2001). To complement this biochemical analysis, we undertook a genetic approach to the analysis of the structure and function of the A20 protein. Here we report the application of clustered charge-to-alanine mutagenesis of the A20 gene. Eight mutant viruses containing altered A20 alleles were isolated using this approach; two of these, tsA20-6 and tsA20-ER5, have tight temperature- sensitive phenotypes. At the nonpermissive temperature, neither virus forms macroscopic plaques and the yield of infectious virus is <1% of that obtained at the permissive temperature. Both viruses show a profound defect in the accumulation of viral DNA at the nonpermissive temperature, although both the A20 protein and DNA polymerase accumulate to wild-type levels. Cytoplasmic extracts prepared from cells infected with the tsA20 viruses show a defect in processive polymerase activity; they are unable to direct the formation of RFII product using a singly primed M13 template. In sum, these data indicate that the A20 protein plays an essential role in the viral life cycle and that viruses with A20 lesions exhibit a DNA phenotype that is correlated with a loss in processive polymerase activity as assayed in vitro. The vaccinia virus A20 protein can, therefore, be considered a new member of the family of proteins (E9, B1, D4, and D5) with essential roles in vaccinia virus DNA replication.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	American Society for Microbiology
ISSN:	0022-538X
Date of First Compliant Deposit:	6 August 2018
Date of Acceptance:	10 September 2001
Last Modified:	11 May 2023 11:51
URI:	https://orca.cardiff.ac.uk/id/eprint/113893

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