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J. Biol. Chem., Vol. 282, Issue 15, 10865-10872, April 13, 2007

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Knockdown of DNA Ligase IV/XRCC4 by RNA Interference Inhibits Herpes Simplex Virus Type I DNA Replication^*

From the Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology, Göteborg University, SE-405 30 Göteborg, Sweden

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.

Received for publication, December 27, 2006 , and in revised form, February 9, 2007.

^* This work was supported by Swedish Cancer Society Grant 2552-B03-17XAC and Swedish Research Council Grant K2005-03X-14255-04A. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1 and S2.

¹ To whom correspondence should be addressed: Institute of Biomedicine, Dept. of Medical Biochemistry and Cell Biology, Göteborg University, Box 440, SE-405 30 Göteborg, Sweden. Tel.: 46-31-7863486; Fax: 46-31-416108; E-mail: per.elias{at}medkem.gu.se.

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