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

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.

The role of the 5′ untranslated regions of Potyviridae in translation

“…Due to the lack of information or limitation in the experimental designs, there is still a major gap in knowledge for the role of the 5 UTRs of most Potyviridae in translation. The VPg has not been reported to be involved in translation for Picornaviridae (Langereis et al, 2014). Note the atypically long 5 untranslated region of the newly emerged virus of wheat, Triticum mosaic virus (Family Potyviridae, genus Poacevirus) with an atypical higher GC content (42.1%) and G, determined by mFOLD prediction of −205.7 kcal/mol, which would suggest stable secondary structures.…”

“…Moreover, potyviruses differ from picornaviruses in that their VPg proteins are several fold larger (picornaviruses ∼2-3 kDa, potyviruses ∼20-23 kDa) and are presumably involved in translation, or at least interact with the cap-binding factors (Wang and Krishnaswamy, 2012). The VPg seems to be dispensable for animal picornavirus translation (Langereis et al, 2014).…”

In vitro labeling strategies for in cellulo fluorescence microscopy of single ribonucleoprotein machines

“…Ideally, these methods should require few steps; are of high efficiency and yield; and use gentle, commercially available, and noncytotoxic materials. Diverse fluorescent labeling methods of endogenous RNAs are available to meet these goals . Many of these methods are plagued by issues relating to limited dye permeability through the cell membrane, nonspecific dye binding, cytotoxicity, necessary modifications to the genome, high‐molecular‐weight RNA extensions, and high intrinsic background; hence, they are often impractical for monitoring low‐abundance and short transcripts .…”

Allosteric inhibitors of Coxsackie virus A24 RNA polymerase

“…In EV, VPg remains bound to the 5’ end of the viral RNA 16 , although it may be removed at some phase of replication 56 . Here, we have shown that the previously identified binding site for VPg on the surface of the CVA24 3D pol (Figure 1 & 2) is, indeed, an allosteric site, as compounds selected to bind there interfere with both uridylylation and subsequent elongation to VPgpolyU (Figures 3 & 5).…”