Propensity of a picornavirus polymerase to slip on potyvirus-derived transcriptional slippage sites.

Hazel Stewart,Allan Olspert,Benjamin G. Butt,Andrew E. Firth

doi:10.1099/jgv.0.001189

Abstract

The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to ‘slip’ during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler’s murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase ‘slips’ at the sequences U6–7 and A6–7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.

Highlights

The substitution rates of viral polymerases have been studied extensively
Polymerase stuttering or slippage can occur within coding sequences to produce populations of transcripts with altered coding capacity, where nucleotide insertions or deletions allow access to alternative open reading frames
By performing infections at an MOI of 0.1, we expected to purge any virus genomes that were defective as a result of slippage occurring during T7 transcription or during virus replication following transfection, so that the ‘Cell I’ and ‘Virus 2’ insertion and deletion data should reflect the viral RNA-dependent RNA polymerase (RdRp) slippage rates

Summary

Introduction

The substitution rates of viral polymerases have been studied extensively. less is known about the tendency of these enzymes to ‘slip’ during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. By performing infections at an MOI of 0.1, we expected to purge any virus genomes that were defective as a result of slippage occurring during T7 transcription or during virus replication following transfection, so that the ‘Cell I’ and ‘Virus 2’ insertion and deletion data should reflect the viral RdRp slippage rates.

Results

Conclusion