Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles
Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-05-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/5/546 |
| _version_ | 1827716821920251904 |
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| author | Edward I. Patterson Kamil Khanipov Daniele M. Swetnam Samantha Walsdorf Tiffany F. Kautz Saravanan Thangamani Yuriy Fofanov Naomi L. Forrester |
| author_facet | Edward I. Patterson Kamil Khanipov Daniele M. Swetnam Samantha Walsdorf Tiffany F. Kautz Saravanan Thangamani Yuriy Fofanov Naomi L. Forrester |
| author_sort | Edward I. Patterson |
| collection | DOAJ |
| description | Mutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance. |
| first_indexed | 2024-03-10T19:48:11Z |
| format | Article |
| id | doaj.art-f5ac04f1d4f84622a72bbb68c82c49e5 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T19:48:11Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-f5ac04f1d4f84622a72bbb68c82c49e52023-11-20T00:37:39ZengMDPI AGViruses1999-49152020-05-0112554610.3390/v12050546Measuring Alphavirus Fidelity Using Non-Infectious Virus ParticlesEdward I. Patterson0Kamil Khanipov1Daniele M. Swetnam2Samantha Walsdorf3Tiffany F. Kautz4Saravanan Thangamani5Yuriy Fofanov6Naomi L. Forrester7Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USADepartment of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Pharmacology and Toxicology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USADepartment of Pathology, University of Texas Medical Branch, Galveston, TX 77555, USAMutations are incorporated into the genomes of RNA viruses at an optimal frequency and altering this precise frequency has been proposed as a strategy to create live-attenuated vaccines. However, determining the effect of specific mutations that alter fidelity has been difficult because of the rapid selection of the virus population during replication. By deleting residues of the structural polyprotein PE2 cleavage site, E3Δ56-59, in Venezuelan equine encephalitis virus (VEEV) TC-83 vaccine strain, non-infectious virus particles were used to assess the effect of single mutations on mutation frequency without the interference of selection that results from multiple replication cycles. Next-generation sequencing analysis revealed a significantly lower frequency of transversion mutations and overall mutation frequency for the fidelity mutants compared to VEEV TC-83 E3Δ56-59. We demonstrate that deletion of the PE2 cleavage site halts virus infection while making the virus particles available for downstream sequencing. The conservation of the site will allow the evaluation of suspected fidelity mutants across alphaviruses of medical importance.https://www.mdpi.com/1999-4915/12/5/546alphavirusarbovirusfidelity mutantsmutation frequency |
| spellingShingle | Edward I. Patterson Kamil Khanipov Daniele M. Swetnam Samantha Walsdorf Tiffany F. Kautz Saravanan Thangamani Yuriy Fofanov Naomi L. Forrester Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles Viruses alphavirus arbovirus fidelity mutants mutation frequency |
| title | Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles |
| title_full | Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles |
| title_fullStr | Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles |
| title_full_unstemmed | Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles |
| title_short | Measuring Alphavirus Fidelity Using Non-Infectious Virus Particles |
| title_sort | measuring alphavirus fidelity using non infectious virus particles |
| topic | alphavirus arbovirus fidelity mutants mutation frequency |
| url | https://www.mdpi.com/1999-4915/12/5/546 |
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