Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus
| Autor: | Amit Das, Madhuparna Bose, Madhurima Roy, Anirban Kundu, Poulomi Biswas, Soumita Dutta, Ananta K. Ghosh, Pradeep Gautam |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
viruses Termination factor RNA-dependent RNA polymerase Biology Viral Proteins 03 medical and health sciences chemistry.chemical_compound Protein Domains Transcription (biology) Virology RNA polymerase Escherichia coli RNA polymerase I Polymerase RNA General Medicine RNA-Dependent RNA Polymerase Molecular biology Nucleopolyhedroviruses 030104 developmental biology chemistry RNA editing biology.protein RNA Viral Protein Binding |
| Zdroj: | Archives of Virology. 162:2727-2736 |
| ISSN: | 1432-8798 0304-8608 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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