Evidence for functional significance of the permuted C motif in Co2+-stimulated RNA-dependent RNA polymerase of infectious bursal disease virus.
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 SO4(2-) anions, the former was a stronger stimulant for RdRp. When cell-culture medium was supplemented with 50 microM 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.
Letzel
Tobias
T
Institutes of Molecular Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.
Mundt
Egbert
E
Institutes of Molecular Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel Riems, Germany.
Gorbalenya
Alexander E
AE
Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300RC Leiden, The Netherlands.
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003035:Cobalt / Q000502:physiology; D006224:Cricetinae; D007243:Infectious bursal disease virus / Q000201:enzymology / Q000235:genetics*; D007668:Kidney; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry; D012367:RNA, Viral / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics
D010446:Peptide Fragments; D012367:RNA, Viral; D014764:Viral Proteins; D003035:Cobalt; D012324:RNA-Dependent RNA Polymerase