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.
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.
Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584 CL, The Netherlands, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, 6500 HB, The Netherlands, Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA and Leiden Institute of Chemistry, Leiden University, Bioorganic Synthesis Leiden, 2300 RA, The Netherlands.
Martijn A
MA
Langereis
Qian
Q
Feng
Frank H T
FH
Nelissen
Richard
R
Virgen-Slane
Gerbrand J
GJ
van der Heden van Noort
Sonia
S
Maciejewski
Dmitri V
DV
Filippov
Bert L
BL
Semler
Floris L
FL
van Delft
Frank J M
FJ
van Kuppeveld
D057930:Click Chemistry; D004770:Enterovirus / Q000235:genetics; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010849:Picornaviridae / Q000235:genetics* / Q000502:physiology; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry*; D014764:Viral Proteins / Q000737:chemistry; D014779:Virus Replication
D010455:Peptides; C034477:RNA primers; D012367:RNA, Viral; C520979:VPg protein, poliovirus; D014764:Viral Proteins; D012313:RNA