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Journal of Virology, April 2005, p. 4691-4699, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4691-4699.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Substitution in Rous Sarcoma Virus Integrase That Separates Its Two Biologically Relevant Enzymatic Activities

Wesley M. Konsavage Jr.,1 Stephen Burkholder,2 Malgorzata Sudol,2 Amy L. Harper,1 and Michael Katzman1,2*

Department of Microbiology and Immunology,1 Department of Medicine, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania2

Received 3 September 2004/ Accepted 28 November 2004

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "–2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of –2 nicks to –3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

* Corresponding author. Mailing address: Department of Medicine, Division of Infectious Diseases, Penn State University College of Medicine, The Milton S. Hershey Medical Center, P.O. Box 850, Mail Code H036, Hershey, PA 17033-0850. Phone: (717) 531-8881. Fax: (717) 531-4633. E-mail: mkatzman{at}psu.edu.

Journal of Virology, April 2005, p. 4691-4699, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4691-4699.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Diamond, T. L., Bushman, F. D. (2005). Division of Labor within Human Immunodeficiency Virus Integrase Complexes: Determinants of Catalysis and Target DNA Capture. J. Virol. 79: 15376-15387 [Abstract] [Full Text]  


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