Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor
Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-04-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/14/4/836 |
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| author | Cesar D. Urrutia Gustavo Romay Brian D. Shaw Jeanmarie Verchot |
| author_facet | Cesar D. Urrutia Gustavo Romay Brian D. Shaw Jeanmarie Verchot |
| author_sort | Cesar D. Urrutia |
| collection | DOAJ |
| description | Plant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species. |
| first_indexed | 2024-03-09T10:27:53Z |
| format | Article |
| id | doaj.art-240e0c46932c4ba19d18ebf84481fa03 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-09T10:27:53Z |
| publishDate | 2022-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-240e0c46932c4ba19d18ebf84481fa032023-12-01T21:31:17ZengMDPI AGViruses1999-49152022-04-0114483610.3390/v14040836Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing SuppressorCesar D. Urrutia0Gustavo Romay1Brian D. Shaw2Jeanmarie Verchot3Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77845, USADepartment of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77845, USADepartment of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77845, USADepartment of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77845, USAPlant infecting emaraviruses have segmented negative strand RNA genomes and little is known about their infection cycles due to the lack of molecular tools for reverse genetic studies. Therefore, we innovated a rose rosette virus (RRV) minireplicon containing the green fluorescent protein (GFP) gene to study the molecular requirements for virus replication and encapsidation. Sequence comparisons among RRV isolates and structural modeling of the RNA dependent RNA polymerase (RdRp) and nucleocapsid (N) revealed three natural mutations of the type species isolate that we reverted to the common species sequences: (a) twenty-one amino acid truncations near the endonuclease domain (named delA), (b) five amino acid substitutions near the putative viral RNA binding loop (subT), and (c) four amino acid substitutions in N (NISE). The delA and subT in the RdRp influenced the levels of GFP, gRNA, and agRNA at 3 but not 5 days post inoculation (dpi), suggesting these sequences are essential for initiating RNA synthesis and replication. The NISE mutation led to sustained GFP, gRNA, and agRNA at 3 and 5 dpi indicating that the N supports continuous replication and GFP expression. Next, we showed that the cucumber mosaic virus (CMV strain FNY) 2b singularly enhanced GFP expression and RRV replication. Including agRNA2 with the RRV replicon produced observable virions. In this study we developed a robust reverse genetic system for investigations into RRV replication and virion assembly that could be a model for other emaravirus species.https://www.mdpi.com/1999-4915/14/4/836plant virusbunyavirusvirion assemblyvirus replicationenvelope glycoproteinsminireplicon |
| spellingShingle | Cesar D. Urrutia Gustavo Romay Brian D. Shaw Jeanmarie Verchot Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor Viruses plant virus bunyavirus virion assembly virus replication envelope glycoproteins minireplicon |
| title | Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor |
| title_full | Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor |
| title_fullStr | Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor |
| title_full_unstemmed | Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor |
| title_short | Advancing the Rose Rosette Virus Minireplicon and Encapsidation System by Incorporating GFP, Mutations, and the CMV 2b Silencing Suppressor |
| title_sort | advancing the rose rosette virus minireplicon and encapsidation system by incorporating gfp mutations and the cmv 2b silencing suppressor |
| topic | plant virus bunyavirus virion assembly virus replication envelope glycoproteins minireplicon |
| url | https://www.mdpi.com/1999-4915/14/4/836 |
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