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J. Biol. Chem., Vol. 279, Issue 15, 14509-14519, April 9, 2004

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Attenuation of HIV-1 Replication in Primary Human Cells with a Designed Zinc Finger Transcription Factor^*

David J. Segal, João Gonçalves¶, Scott Eberhardy, Christina H. Swan||, Bruce E. Torbett||, Xuelin Li, and Carlos F. Barbas, III**

From the The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry and the ^||Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the ^¶URIA-Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, 1649-019 Lisboa, Portugal

Small molecule inhibitors of human immunodeficiency virus, type 1 (HIV-1) have been extremely successful but are associated with a myriad of undesirable effects and require lifelong daily dosing. In this study we explore an alternative approach, that of inducing intracellular immunity using designed, zinc finger-based transcription factors. Three transcriptional repression proteins were engineered to bind sites in the HIV-1 promoter that were expected to be both accessible in chromatin structure and highly conserved in sequence structure among the various HIV-1 subgroups. Transient transfection assays identified one factor, KRAB-HLTR3, as being able to achieve 100-fold repression of an HIV-1 promoter. Specificity of repression was demonstrated by the lack of repression of other promoters. This factor was further shown to repress the replication of several HIV-1 viral strains 10- to 100-fold in T-cell lines and primary human peripheral blood mononuclear cells. Repression was observed for at least 18 days with no significant cytotoxicity. Stable T-cell lines expressing the factor also do not show obvious signs of cytotoxicity. These characteristics present KRAB-HLTR3 as an attractive candidate for development in an intracellular immunization strategy for anti-HIV-1 therapy.

Received for publication, January 13, 2004

^* This work was supported by National Institutes of Health Grants GM065059 and R01 AI49165. 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 nucleotide sequence(s) reported in this paper has been submitted to GenBank^TM/EBI Data Bank with the accession number(s) AY518586, AY518587, and AY518588.

Present address: Dept. of Pharmacology and Toxicology, University of Arizona, Tucson, AZ 85721.

^** To whom correspondence should be addressed: The Scripps Research Institute, BCC-550, North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-9098; Fax: 858-784-2583; E-mail: carlos{at}scripps.edu.

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