A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene
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 v...
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MDPI AG
2020-06-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/6/660 |
| _version_ | 1797564727085236224 |
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| author | Tiffany F. Kautz Elizabeth Jaworski Andrew Routh Naomi L. Forrester |
| author_facet | Tiffany F. Kautz Elizabeth Jaworski Andrew Routh Naomi L. Forrester |
| author_sort | Tiffany F. Kautz |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T19:02:04Z |
| format | Article |
| id | doaj.art-3d406cbfae574e219fdce0ec57317b2c |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T19:02:04Z |
| publishDate | 2020-06-01 |
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| series | Viruses |
| spelling | doaj.art-3d406cbfae574e219fdce0ec57317b2c2023-11-20T04:20:31ZengMDPI AGViruses1999-49152020-06-0112666010.3390/v12060660A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter GeneTiffany F. Kautz0Elizabeth Jaworski1Andrew Routh2Naomi L. Forrester3Institute for Human Infections and Immunity, Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Biochemistry and Molecular Biology, University of Medical Branch, Galveston, TX 77555-0645, USADepartment of Biochemistry and Molecular Biology, University of Medical Branch, Galveston, TX 77555-0645, USAInstitute for Human Infections and Immunity, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USAReporter 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.https://www.mdpi.com/1999-4915/12/6/660arbovirusalphavirusrecombinationsequencingfidelity |
| spellingShingle | Tiffany F. Kautz Elizabeth Jaworski Andrew Routh Naomi L. Forrester A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene Viruses arbovirus alphavirus recombination sequencing fidelity |
| title | A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene |
| title_full | A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene |
| title_fullStr | A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene |
| title_full_unstemmed | A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene |
| title_short | A Low Fidelity Virus Shows Increased Recombination during the Removal of an Alphavirus Reporter Gene |
| title_sort | low fidelity virus shows increased recombination during the removal of an alphavirus reporter gene |
| topic | arbovirus alphavirus recombination sequencing fidelity |
| url | https://www.mdpi.com/1999-4915/12/6/660 |
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