TRENDS in Immunology Vol.23 No.12 December 2002 http://immunology.trends.com 1471-4906/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4906(02)02328-1 559 Research Update Research News Double-stranded RNA-mediated interference (RNAi) induces sequence-specific post-transcriptional gene silencing and has emerged as a powerful tool to silence gene expression in multiple organisms. In mammalian cells, duplexes of 21 nucleotide RNAs, known as short-interfering RNAs (siRNAs), efficiently inhibit gene expression. Recent research demonstrates the general use of siRNAs to specifically inhibit HIV-1 replication by targeting viral or cellular genes. Importantly, RNAi opens a new avenue for gene-based therapeutics. Published online: 14 October 2002 Therapeutic options for combating HIV continue to expand with the development of new drugs and new strategies for their use. The management of HIV-1 infected patients has become increasingly complex. The emergence of drug resistance and the growing recognition of the long-term toxicity of anti-retroviral agents justify a continued effort to develop new antiviral strategies. Intracellular immunization against HIV aims to inhibit virus replication by introducing antiviral genes into the target cell that will then become resistant to infection. Recent findings provide evidence for RNA-mediated interference (RNAi) as a seeding strategy to activate an intracellular host defense mechanism against HIV. RNA interference: the genome’s immune system The phenomenon of RNAi, also referred to as a mechanism of RNA-dependent gene silencing, parallels the immune system because its natural function is the protection of the genome against invasion by mobile genetic elements, such as transposons and viruses (see Forsdyke opinion in this issue). RNAi is usually described as a post-transcriptional gene-silencing (PTGS) phenomenon, in which double-stranded RNA (dsRNA) triggers degradation of homologous mRNA in the cytoplasm. Although this system was first discovered in higher plants [1,2] and later found in the nematode Caenorhabditis elegans [3] it was also observed subsequently in insects, fungi and vertebrates [4], suggesting an ancient evolutionary origin. RNAi has not been found in Archea and prokaryotes and is consequently probably an eukaryotic innovation. The relevance of RNAi as a cellular defense mechanism against intruders was demonstrated by the discovery of plant and insect viruses that encode proteins, which disable PTGS by preventing dsRNA cleavage [2,5]. Whether RNAi has a role in the protection against vertebrate viruses remains to be determined. Similarly, it is unclear if mammalian viruses have developed mechanisms to suppress RNA silencing. Nevertheless, RNAi does not only target dsRNA but also the single-stranded RNAs identical in sequence to the initiator dsRNA, suggesting that RNAi might also act in the regulation of host genes. Mechanism of RNA interference Current data reveal that dsRNA that is homologous in sequence to the silenced gene, serves as the initial trigger of the RNAi mechanism [3] (Fig. 1). In Drosophila, the initial dsRNA is processed by the DICER RNase, a member of the RNase III family of dsRNA-specific endonucleases, into short fragments of 21–25 nts (nucleotides) in length that have 2 or 3 nt 3′ overhangs [1]. These short interfering RNAs (siRNAs) are then incorporated into a dsRNA-inducing silencing complex (RISC) to guide cycles of specific RNA degradation. The RISC contains an endoribonuclease that is probably distinct from DICER [6]. This endoribonuclease uses the sequence encoded by the antisense siRNA strand to find and destroy mRNAs of complementary sequence (Fig. 1). The siRNA, thus, acts as a guide, restricting the ribonuclease to cleavage of only RNAs complementary to RNA interference of HIV replication Miguel Angel Martínez, Bonaventura Clotet and José A. Esté TRENDS in Immunology RISC Viral or transposon dsRNA DICER Active siRNA complex P Target recognition and cleavage P P P P P P P P P Secondary siRNA RNA synthesis by RdRP RISC P P Fig. 1. Model for RNA-mediated interference (RNAi) and silencing. In the RNAi reaction, the cellular RNase III enzyme DICER cleaves the double-stranded RNA (dsRNA) silencing trigger into 21–25-nt RNAs called siRNAs (short-interfering RNA). The siRNAs are then incorporated into a multi-component nuclease, dsRNA-inducing silencing complex (RISC). The antisence siRNA pairs with its cognate mRNA, leading to degradation of target mRNA. Amplification of the silencing signal might be accomplished by siRNAs priming RNA-directed RNA polymerase (RdRP). The RdRP amplification has only been found in Caenorhabditis elegans and plants [29].