The Skaggs Institute
for Chemical Biology

Structure of a Viral Suppressor of RNA Silencing

J.R. Williamson, F. Agnelli, A. Beck, A. Bunner, A. Carmel, J. Chao, B.R. Chapados, E. Debler, S. Edgcomb, M. Hennig, E. Johnson, D. Kerkow, E. Kompfner, K.A. Lehmann, J.H. Lee, R. Muller, H. Reynolds, W. Ridgeway, S.P. Ryder, A. Schneemann, L.G. Scott, E. Sperling, B. Szymczyna, M.W.T. Talkington

The RNA interference or RNA silencing pathway has been an intensively studied phenomenon since it was first discovered several years ago. Introduction of double-stranded RNAs into cells causes specific silencing of gene expression for genes that are complementary to the RNA sequences used. RNA interference is a powerful tool for studying gene expression and has the potential for therapeutic applications. In addition, the existence of the RNA interference pathway has revealed an important and widespread mechanism for gene regulation in a variety of organisms that was only recently appreciated.

Another role for double-stranded RNA is as a signal to cells of a viral infection. Many viruses replicate via a double-stranded RNA intermediate, and one role of the RNA interference pathway seems to be combating viral infection by cleaving viral RNAs that encode viral proteins. Consequently, it is not surprising that viruses have developed counter-defense measures to evade the cellular response to viral infection. A variety of viral proteins have recently been discovered that inhibit the RNA interference pathway, a finding that facilitates replication of virus despite an antiviral defense.

The genome of Flock House virus (FHV) encodes only 4 proteins, one of which is the protein B2, which is a suppressor of the cellular RNA interference pathway. In collaboration with A. Schneemann, Scripps Research, we have biochemically and structurally characterized B2, an accomplishment that has provided some insights into how the virus evades the RNA interference pathway.

B2 protein binds to double-stranded RNA with high affinity but without regard to the sequence. In addition, we found that B2 will bind to double-stranded RNAs of any length and that multiple B2 proteins can bind to longer double-stranded RNAs. Using x-ray crystallography, we solved the structure of B2 bound to an 18-nucleotide double-stranded RNA at 2.6-Å resolution; the structure is shown in 3 orientations in Figure 1.

Fig. 1. X-ray crystal structure of FHV B2 protein bound to double-stranded RNA. The 18-bp duplex RNA is shown as a ribbon with crossbars, and the helical protein is shown with different shades of gray for the 2 copies of the protein in the dimer. Three views (A–C) of the complex are shown that are rotated by 90°.

B2 binds to double-stranded RNA as a dimer in which 2 copies of the protein form a 4-helix bundle. The axis of the helical bundle is roughly parallel to the helical axis of the duplex RNA, and the RNA-protein interface extends over approximately 1.5 turns of the RNA. All of the contacts between the RNA and the protein are made to the sugar-phosphate backbone of the RNA; none are made to the bases, a finding that explains the complete lack of sequence specificity for RNA binding. Two successive minor grooves are contacted by the protein; additional contacts are made to the intervening major groove. The complex has 2-fold symmetry, and the contacts made with 1 minor groove by 1 protein subunit are replicated in the next groove by the symmetry-related protein dimer. The binding of multiple B2 proteins can readily be accommodated, because the protein occupies about one third of the helical circumference. Thus, it is possible to have multiple B2 proteins binding along the double-stranded RNA, essentially coating it with protein.

The RNA interference pathway has several steps where these protein suppressors might act. First, double-stranded RNAs are cleaved by the enzyme Dicer into 21-nucleotide double-stranded RNA fragments. Subsequently, these fragments are incorporated into an RNA silencing complex. This complex can bind to complementary mRNAs and cleave them, as shown in Figure 2. In principle, the RNA interference pathway could be blocked at either of these 2 steps.

Fig. 2. The RNA interference pathway and modes of suppression of RNA silencing. At the center is a schematic for the 2 steps of RNA silencing. First, Dicer cleaves long duplex RNAs into short 21-nucleotide duplex RNAs. Second, these products are incorporated into the RNA silencing complex (RISC). At the left is the structure of the tombusvirus p19 protein bound to 21-nucleotide RNAs; the blunt arrow indicates inhibition of entry into the RISC. At right is the structure of the B2 protein bound to an 18-nucleotide RNA, and the inhibition of the same step is indicated. B2 can also bind multiple copies to longer double-stranded RNAs, a change that inhibits Dicer cleavage, and a model oliomgeric B2 protein complex is shown inhibiting the Dicer cleavage step of the RNA interference pathway.

The tombusvirus protein p19 is another protein that suppresses RNA interference, and this protein binds to the 21-nucleotide fragments that are the products of Dicer cleavage. The structure of p19 bound to a 21-nucleotide double-stranded RNA showed that the protein specifically recognizes the ends of the 21-nucleotide RNAs. Presumably, p19 sequesters the products of Dicer cleavage from incorporation into an RNA silencing complex. The B2 protein can also bind to the 21-nucleotide fragments, and it too can prevent formation of RNA silencing complexes.

However, because B2 can also bind to long double-stranded RNAs, it may also suppress RNA silencing at an earlier stage. Dr. Schneemann and her colleagues have shown that B2 inhibits cleavage of double-stranded RNA by Dicer in vitro. Presumably one of the functions of B2 during FHV infection is to coat the viral genome during replication to preclude entry of the genome into the RNA interference pathway. Thus, B2 suppresses RNA silencing via at least 2 strategies. First, B2 competes for cleavage of double-stranded RNAs by Dicer, and second, any cleaved double-stranded RNAs can still be bound by B2 and prevented from entering the RNA silencing complex. The structure of B2 bound to double-stranded RNA has provided these novel insights into how FHV uses a dual mode of suppressing RNA silencing during replication.

Publications

Chao, J.A., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann A., Williamson, J.R. Dual modes of RNA silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol., in press.

Davis, J.H., Tonelli, M., Scott, L.G., Jaeger, L., Williamson, J.R., Butcher, S.E. RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex. J. Mol. Biol. 351:371, 2005.

Lehmann-Blount, K., Williamson, J.R. Shape-specific recognition of single-stranded RNA by the GLD-1 STAR domain. J. Mol. Biol. 346:91, 2005.

Scott, L.G., Williamson, J.R. The binding interface between Bacillus stearothermophilus ribosomal protein S15 and its 5′ -translational operator mRNA. J. Mol. Biol. 351:280, 2005.

Talkington, M.W., Siuzdak, G., Williamson, J.R. An assembly landscape for the 30S ribosomal subunit. Nature 438:628, 2005.