Nucleic Acids Research Advance Access originally published online on April 13, 2009
Nucleic Acids Research 2009 37(11):3645-3659; doi:10.1093/nar/gkp224
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Nucleic Acids Research, 2009, Vol. 37, No. 11 3645-3659
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Coupled termination/reinitiation for translation of the downstream open reading frame B of the prototypic hypovirus CHV1-EP713
1State Key Lab of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Yuanmingyuan West 2, Beijing 100094, 2Zhejiang Provincial Key Laboratory of Plant Virology, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences Hangzhou 310021, P. R. China and 3Agrivirology Laboratory, Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
*To whom correspondence should be addressed. Tel: +81 86 435 1230; Fax: +81 86 435 1230; Email: nsuzuki{at}rib.okayama-u.ac.jp
Received January 12, 2009. Revised March 6, 2009. Accepted March 23, 2009.
Cryphonectria hypovirus 1 (CHV1), associated with the picorna-like superfamily, infects the chestnut blight fungus and attenuates the virulence of the host fungus. The genomic RNA of the virus has two continuous open reading frames, A and B, separated by the pentanucleotide UAAUG. We present here evidence suggesting that ORF B is translated from genome-sized virus mRNA by a coupled termination/reinitiation mechanism mediated by the pentamer. In the coupled translation, the overlapping UAA and AUG triplets serve as the stop codon of ORF A and the initiator of ORF B, respectively. This was established by the use of a luciferase assay with a basic construct containing the ORF A sequence and the firefly luciferase gene while retaining the pentamer between the two coding sequences. The proportion of ribosomes reinitiating translation after terminating was determined to be 2.5–4.4% by three independent assay systems in fungal and insect cells. Use of a series of mutant constructs identified two sequence elements, the pentamer and the p40 sequence, that affect the efficiency of coupled translation and virus replication. Together, these results provide the first example of coupled translation facilitated by the pentanucleotide UAAUG in the kingdom Fungi. The mechanism by which the preceding p40-coding sequence promotes reinitiation is discussed.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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