Baculovirus expression of the 11 mycoreovirus-1 genome segments and identification of the guanylyltransferase-encoding segment

The type member Mycoreovirus 1 (MyRV-1) of a newly described genus, Mycoreovirus, isolated from a hypovirulent strain 9B21 of the chestnut blight fungus, has a genome composed of 11 dsRNA segments (S1-S11). All of the segments have single ORFs on their capped, positive-sense strands. Infection of insect cells by baculovirus recombinants carrying full-length cDNAs of S1-S11 resulted in overexpression of protein products of the expected sizes, based on their deduced amino acid sequences. This expression system was utilized to identify the S3-encoded protein (VP3) as a guanylyltransferase by an autoguanylylation assay, in which only VP3 was radiolabelled with [α-³²P]GTP. A series of progressive N-terminal and C-terminal deletion mutants was made to localize the autoguanylylation-active site of VP3 to aa residues 133-667. Within this region, a sequence stretch (aa 170-250) with relatively high sequence similarity to homologues of two other mycoreoviruses and two coltiviruses was identified. Site-directed mutagenesis of conserved aa residues revealed that H233, H242, Y243, F244 and F246, but not K172 or K202, play critical roles in guanylyltransferase activities. Together with broader sequence alignments of 'turreted' reoviruses, these results supported the a/ vxxHX₈Hyf/lvf motif, originally noted for orthoreovirus and aquareoviruses, as an active site for guanylyltransferases of viruses within the Orthoreovirus, Aquareovirus, Cypovirus, Oryzavirus, Fijivirus, Coltivirus and Mycoreovirus genera, as well as for the proposed Dinovernavirus genus.