Reassignment of specificities of two cap methyltransferase domains in the reovirus lambda 2 protein

Abstract

Background: The reovirus lambda2 protein catalyzes mRNA capping, that is, addition of a guanosine to the 5' end of each transcript in a 5'-to-5' orientation, as well as transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the N7 atom of the added guanosyl moiety and subsequently to the ribose 2'-O atom of the first template-encoded nucleotide. The structure of the human reovirus core has been solved at 3.6 A resolution, revealing a series of domains that include a putative guanylyltransferase domain and two putative methyltransferase (MTase) domains. It has been suggested that the order of domains in the lambda2 protein corresponds to the order of reactions in the pathway and that the m7G (cap 0) and the 2'-O-ribose (cap 1) MTase activities may be exerted by the MTase 1 and the MTase 2 domains, respectively.

Results: We show that the reovirus MTase 1 domain shares a putative active site with the structurally characterized 2'-O-ribose MTases, including vaccinia virus cap 1 MTase, whereas the MTase 2 domain is structurally similar to glycine N-MTase.

Conclusions: On the basis of our analysis of the structural details we propose that the previously suggested functional assignments of the MTase 1 and MTase 2 domains should be swapped.

Figure 1

Stereoview ribbon diagrams of superimposed structures of the MTase 1 domain (green), VP39 (blue) and RrmJ (magenta). MTase 1 shows elaborations of the common fold similar to those of VP39, which are altogether absent from the RrmJ structure and which are predicted to take part in cap recognition and binding. AdoMet (copied from the RrmJ structure) and the K-D-K-E tetrad of putative catalytic residues are shown in wireframe representation.

Figure 2

Sequence alignment of the predicted human reovirus cap 1 MTase with its counterpart from grass carp reovirus (GCRV) and vaccinia virus (VP39). Residues that are invariant between the reoviruses and those predicted to participate in binding of the AdoMet, the cap moiety, and in catalysis in all cap 1 MTases, are highlighted in black; other conserved residues are highlighted in gray. Motif nomenclature follows [12].

Figure 3

Stereoview of superimposed structures of the MTase 1 domain (ribbon in green, side chains and labels in red) and VP39 (ribbon and labels in blue, side chains in cyan), delineating the proposed binding sites for the guanine moiety and the target ribose 2'-OH. Capped mRNA is shown in orange and the methylated guanine (black label) is sandwiched between Y22 and F180 of VP39. A conformational change of both the protein and the RNA is required for the cluster of conserved aromatic residues of the reovirus MTase to bind the cap structure in a similar manner.

Figure 4

Stereoview of superimposed structures of the MTase 2 domain (green) and GNMT (blue) showing extensive similarities of both domains.

Figure 5

Sequence alignment of the MTase 2 domain with its counterpart from grass carp reovirus. Invariant residues are highlighted in black, conservatively substituted residues are highlighted in gray. Motif nomenclature follows [12].