1. ¹Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
2. ²Viruvation BV Wassenaarseweg 72, Leiden, The Netherlands

Correspondence: Professor Dr B Berkhout, Department of Human Retrovirology, University of Amsterdam, Academic Medical Center K3-110, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands. E-mail: b.berkhout@amc.uva.nl, http://www.berkhoutlab.com

Received 13 December 2005; Revised 20 March 2006; Accepted 22 March 2006; Published online 18 May 2006.

Abstract

Inhibition of virus replication by means of RNA interference has been reported for several important human pathogens, including human immunodeficiency virus type 1 (HIV-1). RNA interference against these pathogens has been accomplished by introduction of virus-specific synthetic small interfering RNAs (siRNAs) or DNA constructs encoding short-hairpin RNAs (shRNAs). Their use as therapeutic antiviral against HIV-1 is limited, because of the emergence of viral escape mutants. In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. Expression of lhRNAs in mammalian cells may result in the synthesis of many siRNAs targeting different viral sequences, thus providing more potent inhibition and reducing the chance of viral escape. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. Our results show that DNA constructs encoding virus-specific lhRNAs are capable of inhibiting HIV-1 production in a sequence-specific manner, without inducing the class I interferon genes.