Abstract
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) is responsible for morbidity of the Indian non-mulberry silkworm, A. mylitta. AmCPV belongs to the family Reoviridae and has 11 double-stranded (ds) RNA genome segments (S1-S11). Segment 2 (S2) encodes a 123-kDa polypeptide with RNA-dependent RNA polymerase (RdRp) activity. To examine the RNA-binding properties of the viral polymerase, the full-length RdRp and its three domains (N-terminal, polymerase and C-terminal domains) were expressed in Escherichia coli BL21 (DE3) cells with hexahistidine and trigger factor tag fused consecutively at its amino terminus, and the soluble fusion proteins were purified. The purified full-length polymerase specifically bound to the 3′ untranslated region (3′-UTR) of a viral plus-sense (+) strand RNA with strong affinity regardless of the salt concentrations, but the isolated polymerase domain of the enzyme exhibited poor RNA-binding ability. Further, the RdRp recognition signals were found to be different from the cis-acting signals that promote minus-sense (-) strand RNA synthesis, because different internal regions of the 3′-UTR of the (+) strand RNA did not effectively compete out the binding of RdRp to the intact 3′-UTR of the (+) strand RNA, but all of these RNA molecules could serve as templates for (-) strand RNA synthesis by the polymerase.
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Acknowledgements
The work was supported by a grant from Department of Science and Technology, Govt. of India (no. SR/SO/BB-0038/2011). AK, MB, MR, SD, PG, and PB thank IIT Kharagpur, CSIR (GOI), DBT (GOI), and ICMR (GOI) for providing research fellowships.
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The authors declare that they have no conflicts of interest. All authors contributed to this work and agreed to its publication. This study represents original work that has not been submitted to any other journal for publication. No human or animal subject was used in this study and therefore, no human or animal ethics approval was required for the completion of this study.
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Kundu, A., Bose, M., Roy, M. et al. Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus. Arch Virol 162, 2727–2736 (2017). https://doi.org/10.1007/s00705-017-3412-3
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DOI: https://doi.org/10.1007/s00705-017-3412-3