@article{mbs:/content/journal/jgv/10.1099/vir.0.82890-0, author = "Letzel, Tobias and Mundt, Egbert and Gorbalenya, Alexander E.", title = "Evidence for functional significance of the permuted C motif in Co2+-stimulated RNA-dependent RNA polymerase of infectious bursal disease virus", journal= "Journal of General Virology", year = "2007", volume = "88", number = "10", pages = "2824-2833", doi = "https://doi.org/10.1099/vir.0.82890-0", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/vir.0.82890-0", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", abstract = "Segment B of bisegmented infectious bursal disease virus (IBDV) encodes virus protein 1 (VP1), possessing RNA-dependent RNA polymerase (RdRp) activity. This multidomain protein includes an RdRp domain with a non-canonical order of three sequence motifs forming the active site: C–A–B. The A–B–C order of the motifs, as found in RdRps of the majority of viruses, was converted by relocation (permutation) of motif C to a C–A–B order. Due to the unusual location and unproven significance, the motif was named ‘C?’. This motif includes an Ala–Asp–Asn tripeptide that replaces the C motif Gly–Asp–Asp sequence, widely considered a hallmark of RdRps. In this study, functional significance of the C? motif was investigated by using purified His-tagged VP1 mutants with either a double replacement (ADN to GDD) or two single-site mutants (ADD or GDN). All mutants showed a significant reduction of RdRp activity in vitro, in comparison to that of VP1. Only the least-affected GDN mutant gave rise to viable, albeit partially impaired, progeny using a reverse-genetics system. Experiments performed to investigate whether the C motif was implicated in the control of metal dependence revealed that, compared with Mn2+ and Mg2+, Co2+ stimulated RdRp unconventionally. No activity was observed in the presence of several divalent cations. Of two Co2+ salts with Cl− and anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 μM Co2+, an increase in IBDV progeny yield was observed. The obtained results provide evidence that the unusual Co2+ dependence of the IBDV RdRp might be linked to the permuted organization of the motif.", }