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Journal of Virology, October 2007, p. 10680-10686, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00950-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Endoribonuclease-Prepared Short Interfering RNAs Induce Effective and Specific Inhibition of Human Immunodeficiency Virus Type 1 Replication{triangledown}

Mireia Gimenez-Barcons,{dagger} Bonaventura Clotet, and Miguel Angel Martinez*

Fundacio irsiCaixa, Universitat Autònoma de Barcelona (UAB), Barcelona, Spain

Received 3 May 2007/ Accepted 15 July 2007

Short interfering RNAs (siRNAs) targeting viral or cellular genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, optimal HIV-1 gene silencing by siRNA requires precise complementarity with most of the target sequence. The emergence of mutations in the targeted gene could lead to rapid viral escape from the siRNA. In the present study, Escherichia coli endoribonuclease III (RNase III) or mammalian Dicer was used to cleave double-stranded RNA into endoribonuclease-prepared siRNA (esiRNA). esiRNAs generate a variety of siRNAs which can efficiently and specifically target multiple sites in the cognate RNA. esiRNAs targeting the region encoding the HIV-1 reverse transcriptase (RT) reduced viral replication by 90%. The inhibition was dose dependent and sequence specific because several irrelevant esiRNAs did not inhibit HIV-1 replication. Importantly, esiRNAs obtained from the prototypic RT sequence of the HXB2 strain and from highly mutated RT sequences showed similar degrees of viral inhibition, suggesting that the heterogeneous population of esiRNAs could overcome individual mismatches in the RT sequence. Finally, esiRNAs generated by Dicer cleavage were five times more potent than those generated by bacterial RNase III digestion. These results show that esiRNAs are potent HIV-1 inhibitors. Moreover, sequence targets do not need to be highly conserved to reach a high level of viral replication inhibition.

* Corresponding author. Mailing address: Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain. Phone: 34-934656374. Fax: 34-934653968. E-mail: mmartinez{at}irsicaixa.es

{triangledown} Published ahead of print on 25 July 2007.

{dagger} Present address: Universitat Pompeu i Fabra (UPF), Barcelona, Spain.

Journal of Virology, October 2007, p. 10680-10686, Vol. 81, No. 19
0022-538X/07/$08.00+0     doi:10.1128/JVI.00950-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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