Structural and Functional Analysis of Methylation and 5′-RNA Sequence Requirements of Short Capped RNAs by the Methyltransferase Domain of Dengue Virus NS5

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTaseDV, is involved in two of four steps required for the formation of the viral mRNA cap 7MeGpppA2′OMe, the guanine-N7 and the adenosine-2′O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2′O-methyltransferase (MTase) activity has been shown on capped 7Me±GpppACn RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 Å to 2.8 Å resolution of NS5MTaseDV in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP–binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of 7MeGpppAC4 and 7MeGpppAC5 RNAs is inhibited in the presence of GTP, 7MeGTP and 7MeGpppA but not by ATP. This particular position of the cap is in accordance with the 2′O-methylation step. A model was generated of a ternary 2′O-methylation complex of NS5MTaseDV, 7MeGpppA and AdoMet. RNA-binding increased when 7Me±GpppAGCn-1 starting with the consensus sequence GpppAG, was used instead of 7Me±GpppACn. In the NS5MTaseDV–GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5′ end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2′O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.