Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Molecular insights into RNA-binding properties of Escherichia coli–expressed RNA-dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

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Publisher
Springer Journals
Copyright
Copyright © 2017 by Springer-Verlag Wien
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-017-3412-3
Publisher site
See Article on Publisher Site

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.

Journal

Archives of VirologySpringer Journals

Published: Jun 6, 2017

References

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