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.
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.
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.
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