Published online 8 December 2006 Nucleic Acids Research, 2007, Vol. 35, No. 1 247–255 doi:10.1093/nar/gkl1022 Use of modified U1 snRNAs to inhibit HIV-1 replication R. Sajic, K. Lee, K. Asai, D. Sakac1, D. R. Branch1, C. Upton2 and A. Cochrane* Department of Medical Genetics and Microbiology and 1Department of Medicine, University of Toronto, Toronto, Ontario, Canada and 2Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada Received August 10, 2006; Revised November 13, 2006; Accepted November 14, 2006 ABSTRACT Control of RNA processing plays a central role in regulating the replication of HIV-1, in particular the 30 polyadenylation of viral RNA. Based on the demonstration that polyadenylation of mRNAs can be disrupted by the targeted binding of modified U1 snRNA, we examined whether binding of U1 snRNAs to conserved 10 nt regions within the terminal exon of HIV-1 was able to inhibit viral structural protein expression. In this report, we demonstrate that U1 snRNAs complementary to 5 of the 15 regions targeted result in significant suppression of HIV-1 protein expression and viral replication coincident with loss of viral RNA. Suppression of viral gene expression is dependent upon appropriate ass- embly of a U1 snRNP particle as mutations of U1 snRNA that affect binding of U1 70K or Sm proteins significantly reduced efficacy. However, constructs lacking U1A binding sites retained significant anti- viral activity. This finding suggests a role for these mutants in situations where the wild-type constructs cause toxic effects. The conserved nature of the sequences targeted and the high efficacy of the con- structs suggests that this strategy has significant potential as an HIV therapeutic. INTRODUCTION As is the case for all retroviruses, replication of HIV-1 is critically dependent upon the controlled processing of its RNA. From a single 9 kb viral transcript, 15 proteins are generated through the process of suboptimal splicing that generates over 30 viral mRNAs (1,2). Control of splicing is achieved in part through the suboptimal nature of the 30 splice sites (ss) present (3–5) as well as the presence of both exon splicing enhancers (ESEs) and exon splicing silencers (ESSs) (5–11). In the course of analyzing the role of the ESE within the terminal exon of HIV-1 RNA, we observed that, in addition to promoting the use of the adjacent 30 ss, the ESE was also required for the 30 end cleavage and polyadenylation of the incompletely spliced viral RNAs (12). Deletion of this ESE resulted in loss of gp160 expression due to a failure to transport the incompletely spliced viral RNA to the cytoplasm (12,13). The correlation of the block to viral RNA export with a reduction in 30 end formation suggested that targeting this step in the viral life cycle may provide an additional strategy to control HIV-1 replication. The formation of the 30 end of RNA polymerase II tran- scripts is determined in large part by two sequences within the RNA; the consensus AAUAAA polyadenylation signal and the downstream enhancing sequence consisting of either a G or G/U-rich region (14,15). In addition to these basic signals, evidence has also emerged for regulation by trans- acting factors, one of which is U1 snRNA. In several systems studied to date [HIV-1 polyadenylation near the start site of transcription, polyadenylation of the late transcripts of bovine papillomavirus (BPV)], interaction of U1 snRNP via base- pairing close to the polyadenylation signal results in either inhibition of the cleavage or polyadenylation portion of the 30 end processing reaction (16–20). More recently, this effect has been exploited to suppress other genes by the expression of variants of U1 snRNA in which the ﬁrst 10 nt have been substituted with a sequence complementary to the gene of interest (21–23). Provided that the targeted sequence is pre- sent within the terminal exon of the gene of interest and not present within secondary structure (i.e. is accessible for base pairing with U1 snRNA), a signiﬁcant inhibition of gene expression can be achieved. To explore the utility of U1 snRNA variants to inhibit HIV-1 gene expression, we identiﬁed highly conserved 10 nt sequences within the terminal exon of the M and O clades of HIV-1 and prepared U1 snRNAs expression vectors with complementarity to 15 of these regions. Several of these con- structs induced a dramatic suppression of the expression of HIV-1 p55/p24 upon cotransfection with a HIV-1 proviral expression vector. Little or no effect on expression of these proteins was detected upon overexpression of U1 snRNAs that had no complementarity to HIV-1 and the anti-HIV-1 constructs had no effect on other co-transfected reporter vec- tors. Combination of anti-HIV constructs leads to an even greater degree of inhibition. The loss of viral protein expres- sion was accompanied by a dramatic decrease in levels of *To whom correspondence should be addressed at Department of Medical Genetics and Microbiology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S-1A8. Tel: +416 978 2500; Fax: +416 978-6885; Email: alan.cochrane@utoronto.ca 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.