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Title: Single-cell mutation rate of turnip crinkle virus (-)-strand replication intermediates

Viruses with single-stranded, positive-sense (+) RNA genomes incur high numbers of errors during replication, thereby creating diversified genome populations from which new, better adapted viral variants can emerge. However, a definitive error rate is known for a relatively few (+) RNA plant viruses, due to challenges to account for perturbations caused by natural selection and/or experimental set-ups. To address these challenges, we developed a new approach that exclusively profiled errors in the (-)-strand replication intermediates of turnip crinkle virus (TCV), in singly infected cells. A series of controls and safeguards were devised to ensure errors inherent to the experimental process were accounted for. This approach permitted the estimation of a TCV error rate of 8.47 X 10−5substitution per nucleotide site per cell infection. Importantly, the characteristic error distribution pattern among the 50 copies of 2,363-base-pair cDNA fragments predicted that nearly all TCV (-) strands were products of one replication cycle per cell. Furthermore, some of the errors probably elevated error frequencies by lowering the fidelity of TCV RNA-dependent RNA polymerase, and/or permitting occasional re-replication of progeny genomes. In summary, by profiling errors in TCV (-)-strand intermediates incurred during replication in single cells, this study provided strong support for a stamping machine mode of replication employed by a (+) RNA virus.

 
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Award ID(s):
1758912
NSF-PAR ID:
10479651
Author(s) / Creator(s):
; ; ; ; ; ;
Editor(s):
Pagán, Israel
Publisher / Repository:
PLoS Pathogens
Date Published:
Journal Name:
PLOS Pathogens
Volume:
19
Issue:
8
ISSN:
1553-7374
Page Range / eLocation ID:
e1011395
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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