Abstract
Reporter genes for RNA viruses are well-known to be unstable due to putative RNA recombination events that excise inserted nucleic acids. RNA recombination has been demonstrated to be co-regulated with replication fidelity in alphaviruses, but it is unknown how recombination events at the minority variant level act, which is important for vaccine and trans-gene delivery design. Therefore, we sought to characterize the removal of a reporter gene by a low-fidelity alphavirus mutant over multiple replication cycles. To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. Short-read RNA sequencing using ClickSeq was performed to determine which regions of the viral genome underwent recombination and how this changed over multiple replication cycles. A rapid removal of the GFP gene was observed, where minority variants in the virus population accumulated small deletions that increased in size over the course of passaging. Eventually, these small deletions merged to fully remove the GFP gene. The removal was significantly enhanced during the passaging of low-fidelity TC-83, suggesting that increased levels of recombination are a defining characteristic of this mutant.
Highlights
RNA viruses are notoriously error-prone due to the lack of an RNA-dependent RNA polymerase (RdRp) proofreading enzyme
It is not well understood how reporter gene removal occurs in virus populations because previous research has typically been limited to the use of low-throughput sequencing methods, generally by Sanger sequencing of a handful of virus clones surrounding a small area of the virus genome
We used ClickSeq to better understand how GFP is removed when inserted into an alphavirus genome under a double subgenomic promoter
Summary
RNA viruses are notoriously error-prone due to the lack of an RNA-dependent RNA polymerase (RdRp) proofreading enzyme (with the unique exception of proofreading exonucleases present in coronavirus genomes). The molecular determinants of RNA recombination are not well-defined, but RNA recombination has been described for viruses such as poliovirus and brome-mosaic virus to arise more frequently in regions with high amounts of RNA secondary structure and AU-rich sequences [9,10]. Other factors, such as sequence homology between RNA strands and altered replication kinetics have been implicated in changing the rate of RNA recombination [9]. We found that a low-fidelity variant of TC-83 [23] removed the GFP reporter gene much more efficiently
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