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Journal of Virology, September 2008, p. 8362-8372, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00900-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differential Requirements of the C Terminus of Nbs1 in Suppressing Adenovirus DNA Replication and Promoting Concatemer Formation{triangledown}

Seema S. Lakdawala,1,2 Rachel A. Schwartz,1,2 Kevin Ferenchak,1 Christian T. Carson,1,2,{dagger} Brian P. McSharry,3 Gavin W. Wilkinson,3 and Matthew D. Weitzman1*

Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California 92037,1 Graduate Program, Division of Biology, University of California, San Diego, California 92093,2 Department of Medical Microbiology, Cardiff University, Cardiff CF14 4XX, United Kingdom3

Received 30 April 2008/ Accepted 12 June 2008

Adenoviruses (Ad) with the early region E4 deleted (E4-deleted virus) are defective for DNA replication and late protein synthesis. Infection with E4-deleted viruses results in activation of a DNA damage response, accumulation of cellular repair factors in foci at viral replication centers, and joining together of viral genomes into concatemers. The cellular DNA repair complex composed of Mre11, Rad50, and Nbs1 (MRN) is required for concatemer formation and full activation of damage signaling through the protein kinases Ataxia-telangiectasia mutated (ATM) and ATM-Rad3-related (ATR). The E4orf3 and E4orf6 proteins expressed from the E4 region of Ad type 5 (Ad5) inactivate the MRN complex by degradation and mislocalization, and prevent the DNA damage response. Here we investigated individual contributions of the MRN complex, concatemer formation, and damage signaling to viral DNA replication during infection with E4-deleted virus. Using virus mutants, short hairpin RNA knockdown and hypomorphic cell lines, we show that inactivation of MRN results in increased viral replication. We demonstrate that defective replication in the absence of E4 is not due to concatemer formation or DNA damage signaling. The C terminus of Nbs1 is required for the inhibition of Ad DNA replication and recruitment of MRN to viral replication centers. We identified regions of Nbs1 that are differentially required for concatemer formation and inhibition of Ad DNA replication. These results demonstrate that targeting of the MRN complex explains the redundant functions of E4orf3 and E4orf6 in promoting Ad DNA replication. Understanding how MRN impacts the adenoviral life cycle will provide insights into the functions of this DNA damage sensor.

* Corresponding author. Mailing address: Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 453-4100, ext. 2037. Fax: (858) 558 7454. E-mail: weitzman{at}salk.edu

{triangledown} Published ahead of print on 18 June 2008.

{dagger} Present address: Becton Dickinson Biosciences, San Diego, CA.

Journal of Virology, September 2008, p. 8362-8372, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00900-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Blackford, A. N., Grand, R. J. A. (2009). Adenovirus E1B 55-Kilodalton Protein: Multiple Roles in Viral Infection and Cell Transformation. J. Virol. 83: 4000-4012 [Full Text]  
  • Karen, K. A., Hoey, P. J., Young, C. S. H., Hearing, P. (2009). Temporal Regulation of the Mre11-Rad50-Nbs1 Complex during Adenovirus Infection. J. Virol. 83: 4565-4573 [Abstract] [Full Text]  


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