Mutagenesis of the murine cytomegalovirus M56 terminase gene

The murine cytomegalovirus (MCMV) M56 is one of three proteins that combine to form the MCMV terminase, required for cleavage and packaging of viral DNA into capsids. Deletion of M56 from a bacterial artificial chromosome (BAC) clone of the MCMV genome was considered lethal, as the mutant BAC failed to reconstitute infectious virus. Reintroduction of M56 at an ectopic locus complemented the deletion, allowing reconstitution of a virus that replicated with wild-type efficiency. However, neither the reintroduction of M56 sequences encoding an N-terminal epitope fusion nor a mutation targeting a region in M56 implicated as an ATPase active site was capable of restoring virus viability. In contrast, a frame shift mutation in M56a, a putative open reading frame that overlaps M56, had no effect on viral replication. We conclude that M56a is dispensable, whereas M56 residues comprising the proposed ATPase active site are critical for terminase function and viral replication.