How viruses sabotage silencing.

The discovery and description of RNA silencing less than a decade ago has spawned a flood of research, revolutionizing the practice of functional genomics and leading to intensive exploration of its potential application to treat numerous diseases. RNA silencing was first noticed in plants when attempts to create transgenic plants that overexpressed a natural gene often had the opposite effect; later it was found to be an evolutionarily conserved defense mechanism against plant RNA viruses and other molecular parasites. Viruses, in turn, have evolved their own counterdefense mechanisms: proteins that suppress RNA silencing, allowing the virus to maintain its invasion of a plant. Until recently, the mechanism behind this suppression of silencing was a mystery, but researchers at the NIEHS and the Agricultural Biotechnology Center in Godollo, Hungary, have begun to unravel how some viruses neutralize silencing, shedding important new light on a complex molecular interaction.

In the 26 December 2003 issue of Cell, NIEHS investigator Tract M. Tanaka Hall, postdoctoral researcher Jeffrey Vargason, and Hungarian colleagues Jozsef Burgyan and Gyorgy Szittly elucidate the nature of viral counterdefense by solving the crystal structure of a known silencing suppressor, the tombusvirus Carnation Italian ringspot virus (CIRV) p19 protein, in complex with a 21-nucleotide small interfering RNA (siRNA), the workhorse bit of nucleic acid that drives the silencing process. The structure of a similar p19 protein found in another tombusvirus was published by Keqiong Ye, Lucy Malinina, and Dinshaw Patel, all of the Memorial Sloan-Kettering Cancer Center, in the 18/25 December 2003 issue of Nature. The slight differences in the structures have allowed researchers to draw further inferences about how a virus can interfere with RNA silencing.

There are two classes of plant siRNAs. The shorter ones, measuring 21-22 nucleotides, are responsible for detecting and destroying molecular invaders. The longer ones, measuring 24-26 nucleotides, are suspected to be more involved with regulating retrotransposons and DNA methylation. The structure of p19 reveals that the protein selectively recognizes silencing siRNAs by measuring their length. Tryptophan residues (Trp39 and Trp42) on the protein act like molecular calipers, forming a so-called stacking interaction with the ends of the end base pairs of the shorter siRNAs. By binding to the silencing siRNAs, the protein in effect sequesters them, rendering them incapable of carrying out their silencing mission and allowing the virus to run rampant within the plant.

The protein can also bind to the longer siRNAs, but much more weakly. "Viruses are smart," says structural biologist Tanaka Hall. "They don't want to kill their hosts too quickly. They want to have time to replicate. So it makes sense that the virus spares processes that might be essential to the plant's survival and not directed against virus invasion."

The team also studied the importance of tryptophan residues to p19's silencing suppression effects by introducing mutations into the p19 coding sequence of the CIRV genome to change one or both of the tryptophan residues. A protein database search had shown that both tryptophans were not absolutely conserved in all identified viral p19 sequences, although all sequences conserved at least one of the tryptophans, with the other residue usually capable of forming a stacking interaction. Their findings suggest that while the virus can at least partially succeed in suppressing silencing with two residues capable of stacking, substituting one with a glycine residue lacking a side chain results in failure of the suppression and recovery of the plant from infection.

p19 has emerged as an important research tool in the quest to refine scientific understanding of how siRNAs and other small RNAs, such as microRNAs, work in planta, in vitro, and in vivo. Unlike silencing siRNAs themselves, which rely on sequence specificity to accomplish their effects, p19 appears to simply measure the siRNAs and neutralize those designed for silencing. This mechanistic revelation will enhance the value of the protein as researchers continue their efforts to clarify the biological roles of small RNAs in gene silencing and other cellular regulatory and developmental processes.

According to RNA silencing research pioneer Phillip Zamore, an associate professor in the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School, Tanaka Hall's structure of the p19 protein also provides great insight into the RNA silencing pathway itself. "All future models of the RNA interference pathway must incorporate a step at which they are vulnerable to siRNA sequestration by p19," he says. "This conclusion is inescapable once one has seen the structure."

Tanaka Hall's group plans to continue to use p19 in its investigations. "We're particularly looking at its role in being able to inhibit microRNA-initiated processes, looking biochemically at how p19 combines with microRNAs," she says. Structural imaging of that relationship could aid in explaining the still poorly understood mechanisms by which microRNAs accomplish their cellular tasks.