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Journal of Virology, March 2006, p. 2873-2883, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.2873-2883.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Immunodeficiency Virus Type 1 Replication with Artificial Transcription Factors Targeting the Highly Conserved Primer-Binding Site

Scott R. Eberhardy,1 Joao Goncalves,2 Sofia Coelho,2 David J. Segal,1,{dagger} Ben Berkhout,3 and Carlos F. Barbas III1*

The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, California 92037,1 The URIA-Centro de Patogenese Molecular, Faculdade de Farmacia, University of Lisbon, 1649-019 Lisbon, Portugal,2 Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands3

Received 30 August 2005/ Accepted 19 December 2005

The human immunodeficiency virus type 1 (HIV-1) primer-binding site (PBS) is a highly conserved region in the HIV genome and represents an attractive target for the development of new anti-HIV therapies. In this study, we designed four artificial zinc finger transcription factors to bind at or adjacent to the PBS and repress transcription from the HIV-1 long terminal repeat (LTR). These proteins bound to the LTR in vivo, as demonstrated by the chromatin immunoprecipitation assay. In transient reporter assays, three of the four proteins repressed transcription of a reporter driven by the HIV-1 LTR. Only one of these proteins, however, designated KRAB-PBS2, was able to prevent virus production when transduced into primary lymphocytes. We observed >90% inhibition of viral replication over the course of several weeks compared to untransduced cells, and no significant cytotoxicity was observed. Long-term exposure of HIV-1 to KRAB-PBS2 induced mutations in the HIV-1 PBS that reduced the effectiveness of the repressor, but these mutations also resulted in decreased rates of viral replication. These results show that KRAB-PBS2 has the potential to be used in antiviral therapy for AIDS patients and might complement other gene-based strategies.

* Corresponding author. Mailing address: The Scripps Research Institute, Department of Molecular Biology, BCC 550, 10050 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-9098. Fax: (858) 784-2583. E-mail: carlos{at}scripps.edu.

{dagger} Present address: University of California, UC Davis Genome Center, 451 E. Health Sciences Dr., Davis, CA 95616.

Journal of Virology, March 2006, p. 2873-2883, Vol. 80, No. 6
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.6.2873-2883.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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