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Journal of Virology, May 2006, p. 4847-4857, Vol. 80, No. 10
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.10.4847-4857.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

African Swine Fever Virus Protein pE296R Is a DNA Repair Apurinic/Apyrimidinic Endonuclease Required for Virus Growth in Swine Macrophages

Modesto Redrejo-Rodríguez, Ramón García-Escudero,{dagger} Rafael J. Yáñez-Muñoz,{ddagger} María L. Salas, and José Salas*

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Received 15 December 2005/ Accepted 21 February 2006

We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the Km and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3'->5' exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.

* Corresponding author. Mailing address: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. Phone: 34 91 497 84 78. Fax: 34 91 497 47 99. E-mail: mlsalas{at}cbm.uam.es.

{dagger} Present address: Cellular and Molecular Biology, CIEMAT, Madrid, Spain.

{ddagger} Present address: Nuclear Biology Group, Department of Medical and Molecular Genetics, King's College London, Guy's Hospital, London, United Kingdom.

Journal of Virology, May 2006, p. 4847-4857, Vol. 80, No. 10
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.10.4847-4857.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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