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

Abstract

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase DV, is involved in two of four steps required for the formation of the viral mRNA cap 7MeGpppA 2′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±GpppAC n 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 NS5MTase DV 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 7MeGpppAC 4 and 7MeGpppAC 5 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 NS5MTase DV, 7MeGpppA and AdoMet. RNA-binding increased when 7Me±GpppAGC n-1 starting with the consensus sequence GpppAG, was used instead of 7Me±GpppAC n . In the NS5MTase DV–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.