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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 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 Mn and Mg, Co stimulated RdRp unconventionally. No activity was observed in the presence of several divalent cations. Of two Co salts with Cl and anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 μM Co, an increase in IBDV progeny yield was observed. The obtained results provide evidence that the unusual Co dependence of the IBDV RdRp might be linked to the permuted organization of the motif.

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2007-10-01
2019-11-14
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vol. , part 10, pp. 2824 – 2833

Oligonucleotides used for cloning procedures

Purification of VP1 from 9 cells and growth analysis of recombinant virus in cell culture

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