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Journal of Virology, February 2006, p. 1863-1873, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1863-1873.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-Inducible Coexpression of a Short Hairpin RNA and Protein

Hoshang J. Unwalla,1 Hai-Tang Li,1 Ingrid Bahner,2 Ming-Jie Li,1 Donald Kohn,2 and John J. Rossi1*

Division of Molecular Biology, Beckman Research Institute of The City of Hope, Duarte, California 91010,1 Division of Research Immunology and Bone Marrow Transplantation, Childrens Hospital, Los Angeles, California 900272

Received 15 August 2005/ Accepted 17 November 2005

We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.

* Corresponding author. Mailing address: Department of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010. Phone: (626) 301-8360. Fax: (626) 301-8271. E-mail: jrossi{at}coh.org.

Journal of Virology, February 2006, p. 1863-1873, Vol. 80, No. 4
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.4.1863-1873.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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