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Evidence for functional significance of the permuted C motif in Co²⁺-stimulated RNA-dependent RNA polymerase of infectious bursal disease virus

Egbert Mundt^1,

¹ Institutes of Molecular Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany
² Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300RC Leiden, The Netherlands

Correspondence
Egbert Mundt
emundt{at}uga.edu

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.

Present address: The University of Georgia, Poultry Diagnostic and Research Center, College of Veterinary Medicine, 953 College Station Rd, Athens, GA 30602, USA.

Supplementary methods and a supplementary table showing oligonucleotides used for cloning procedures are available with the online version of this paper.