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Modification of picornavirus genomic RNA using 'click' chemistry shows that unlinking of the VPg peptide is dispensable for translation and replication of the incoming viral RNA.

Nucleic acids research (2013-11-19)
Martijn A Langereis, Qian Feng, Frank H T Nelissen, Richard Virgen-Slane, Gerbrand J van der Heden van Noort, Sonia Maciejewski, Dmitri V Filippov, Bert L Semler, Floris L van Delft, Frank J M van Kuppeveld
ABSTRACT

Picornaviruses constitute a large group of viruses comprising medically and economically important pathogens such as poliovirus, coxsackievirus, rhinovirus, enterovirus 71 and foot-and-mouth disease virus. A unique characteristic of these viruses is the use of a viral peptide (VPg) as primer for viral RNA synthesis. As a consequence, all newly formed viral RNA molecules possess a covalently linked VPg peptide. It is known that VPg is enzymatically released from the incoming viral RNA by a host protein, called TDP2, but it is still unclear whether the release of VPg is necessary to initiate RNA translation. To study the possible requirement of VPg release for RNA translation, we developed a novel method to modify the genomic viral RNA with VPg linked via a 'non-cleavable' bond. We coupled an azide-modified VPg peptide to an RNA primer harboring a cyclooctyne [bicyclo[6.1.0]nonyne (BCN)] by a copper-free 'click' reaction, leading to a VPg-triazole-RNA construct that was 'non-cleavable' by TDP2. We successfully ligated the VPg-RNA complex to the viral genomic RNA, directed by base pairing. We show that the lack of VPg unlinkase does not influence RNA translation or replication. Thus, the release of the VPg from the incoming viral RNA is not a prerequisite for RNA translation or replication.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
GenElute Mammalian Total RNA Miniprep Kit, sufficient for 70 purifications
Sigma-Aldrich
GenElute Mammalian Total RNA Miniprep Kit, sufficient for 350 purifications
Sigma-Aldrich
GenElute Mammalian Total RNA Miniprep Kit, sufficient for 10 purifications

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