Clearance of viral pathogens from a variety of hosts is often the result of the destruction of infected cells. However, mechanisms that enable cell survival following infection have been described. Studies in plants suggest that one such mechanism may be based on RNA-induced gene silencing, now referred to as RNA silencing or RNA interference (RNAi). This ancient pathway is conserved among species from different kingdoms (fungi, animals and plants) and controls gene expression at the transcriptional and post-transcriptional level.

The post-transcriptional activity of the RNAi machinery to degrade cytoplasmic RNA in a sequence specific manner is key to its antiviral function. Biochemical studies indicate that RNAi proceeds via a two-step mechanism. In the first step, long dsRNAs are produced locally or taken up by the cells and cleaved by the RNase III-like nuclease Dicer, which generates 21-23 nucleotide duplex RNAs (siRNA). In the second step, siRNAs are incorporated into a multicomponent nuclease complex, the RNA-induced silencing complex (RISC). The antisense strand of the duplex serves as a guide that directs RISC to recognize and cleave cognate mRNA. Because of the extreme specificity and efficiency of the RNAi machinery, this mechanism has the ability to clear plant and mammalian cells of viral infections.

The RNAi process is very efficient: a few dsRNA molecules can trigger inactivation of a continuously transcribed target mRNA for prolonged periods of time. The RNAi-induced inactivation persists through cell division and in some organisms can spread to untreated cells and tissues; when the RNAi spreads into germ line cells it can even be inherited by subsequent generations. Although amplification and spreading of RNA silencing have been demonstrated in plants, nematodes and in vitro in Drosophila extracts, they have not been yet observed in mammalian systems.

A role for RNAi pathways in the regulation of endogenous genes was suggested through the analysis of plants and animals containing dysfunctional RNAi components. For example, mutations in a component of RISC cause pleiotropic developmental abnormalities in Arabidopsis. A mutation in the Arabidopsis Dicer ortholog causes defects in leaf development and overproliferation of floral meristems. Mutations in Argonaute family members in Drosophila impact normal development with drastic effects in neuronal development and mutations in another RISC component, piwi, result in defects in both germline stem-cell proliferation and maintenance. The emerging view is that RNA silencing is part of a sophisticated network of interconnected pathways for cellular defense (pathogen resistance and stabilization of mobile genetic elements), RNA surveillance (chromatin remodeling, genome organization and stability) and development. In this proposal we will investigate the role of RNAi in the progress of viral infection and the possibility of using the system as a novel antiviral therapeutic approach.

There is a good deal of genetic support for the importance of RNAi in antiviral defense. In plants, RNAi is clearly involved in the response to viruses: Arabidopsis strains defective in post transcriptional gene silencing are more susceptible to virus infections, and a substantial number of plant viruses encode proteins that counter silencing. RNA interference (RNAi) is also a major antiviral defense mechanism in arthropods. We have shown that Drosophila melanogaster strains defective in RNAi core components, Dicer2 and AGO-2, are highly hypersensitive to infection, resulting in a 1000-fold increase in virus production. In turn, viruses have evolved mechanism to suppress RNAi. A central question to the concept of an RNA-based immune system is whether the process relies on a cell-autonomous or systemic mechanism. We investigated this problem by focusing on a critical aspect of the putative systemic spread process: dsRNA uptake. Drosophila S2 cells can efficiently take up dsRNA. We combined biochemical, pharmacological, cell biological and genomic approaches to examine the dsRNA uptake mechanism in Drosophila. Our experiments indicate that exogenous dsRNA enters the RNAi pathway via scavenger receptor-mediated endocytosis. We also identified several genes involved in the sub-cellular localization of dsRNA critical for silencing. Notably, mutant flies defective in genes identified in our Drosophila S2 cell screen are hypersensitive to virus infection, indicating that RNAi uptake is essential in the process of antiviral defense. A key question that remains unanswered is whether RNAi has a role as a natural antiviral defense in mammals.