© National Academy of Sciences, USA
Viroid replication generates dsRNA intermediates, which are processed by Dicer into 21- to 25-nucleotide siRNAs. These siRNAs are then incorporated into siRNA – ribonuclease complexes (RISC). If the siRNA sequences significantly match host mRNAs, RISC may target them for degradation leading to disease symptoms. RISC can also target the viroid, forcing it to evolve and to adopt and maintain an RNA silencing-resistant structure. (From M.-B. Wang et al.,
There is growing evidence that small RNA's believed to play an antiviral defense role in many organisms, may be acting as doubleagents. In March 2004, an international team of scientists reported that viroids, small infectious particles of naked RNA, may be employing RNA-silencing machinery to work their damage.1 Several weeks later, another international team announced the discovery of microRNAs in the Epstein-Barr virus (EBV), suggesting that miRNA-mediated gene suppression might play a role in animal-virus pathogenicity.2 Many researchers say this is just the beginning.
"In the past, all of these small RNAs were totally overlooked because they are so small in size. If they got any attention at all, they were considered unspecific degradation products. Now, we are starting to understand that they represent a sort of cross-talk between viral or viroidal sequences and endogenous genes," says Martin Tabler, Institute of Molecular Biology and Biotechnology, Crete.
EBV, a large DNA virus responsible for mononucleosis and a number of other diseases, including Burkitt lymphoma, is tough to beat; it hides from the human immune system and infects for life. Rockefeller University's Sebastien Pfeffer, lead author on the EBV study, says that he and his collaborators were looking for evidence that animals defend against viral infection using the same kind of posttranscriptional silencing pathways that plants, insects, and other organisms use. But instead of antiviral RNA activity, they discovered viral miRNA activity. According to Pfeffer, the data suggest that miRNAs could be involved in tumor formation and may explain how EBV hides so well. They also give scientists reason to look for miRNAs in other viruses. Says Pfeffer: "miRNAs are found in practically every eukaryotic organism. Other viruses could have them too."
RNA SILENCING: A WEAPON OF CHOICE?
Viroids are single RNA molecules that have no protective protein coat and do not encode a single protein. While not all viroids inflict damage, some do so severely. A potato spindle tuber viroid (PSTVd) infection can reduce yields by 50% or more. As Ricardo Flores of the Valencia Polytechnical University, Spain, explains, "Viruses can induce disease by means of proteins encoded in their genomes, but viroids cannot. So the question is, how?"
For years, most researchers believed that the viroids induced disease by interrupting an unknown host factor (i.e., protein) and thereby disrupting normal cell functioning. This hypothesis was proposed in the mid-1980's, when scientists identified a region of the viroid genome, the "virulence-modulating" region, whose sequence, when altered, affected virulence. But the mechanistic details of this viroid-host interaction remained a mystery. In 2001 it was suggested that, rather than interacting directly with the host, perhaps viroids use small regulatory RNAs to influence host gene expression.
In March 2004, Ming-Bo Wang and colleagues from the Commonwealth Scientific and Industrial Research Organization in Canberra, Australia, provided the first experimental evidence suggesting that, similar to EBV, the key to viroid pathogenicity may indeed be RNA silencing, but in this case, siRNA-mediated, not miRNA-mediated. The investigators found that engineered tomato plants expressing viroid-derived, noninfectious hairpin RNA had symptoms mimicking those of viroid infection. Hairpin RNA is a key player in the RNA silencing pathway: When cleaved by a Dicer enzyme, it generates siRNAs, which in turn guide RISC complexes to degrade target RNA. In this case, the target RNA hasn't been identified, and Wang says this is a necessary next step.
"This is one of the most exciting things in the field," says Flores. "But more evidence is needed before deciding which of the two alternative hypotheses is correct. If you want to prove this hypothesis, you have to identify which host mRNA is degraded." For Tabler, the results are "surprising and actually a bit puzzling." They raise interesting questions about how nuclear-replicating viroids such as PSTVd exploit RNA-silencing machinery, which is located in the cytoplasm.
Future findings aside, both studies nonetheless confirm the ubiquitous nature of small RNAs and point to an additional, pathogenic function for RNA regulatory mechanisms. Says Andrew Ellington, University of Texas: "It's exciting to see the small RNA world sow its oats."
Leslie A. Pray