Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity
Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs effi...
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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/1999-4915/12/5/568 |
| _version_ | 1797567293590339584 |
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| author | Shuohui Liu Rebecca Kaddis Maldonado Tiffiny Rye-McCurdy Christiana Binkley Aissatou Bah Eunice C. Chen Breanna L. Rice Leslie J. Parent Karin Musier-Forsyth |
| author_facet | Shuohui Liu Rebecca Kaddis Maldonado Tiffiny Rye-McCurdy Christiana Binkley Aissatou Bah Eunice C. Chen Breanna L. Rice Leslie J. Parent Karin Musier-Forsyth |
| author_sort | Shuohui Liu |
| collection | DOAJ |
| description | Retroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions. |
| first_indexed | 2024-03-10T19:39:43Z |
| format | Article |
| id | doaj.art-6aa298b580df42dcb1f56745c37972bd |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T19:39:43Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-6aa298b580df42dcb1f56745c37972bd2023-11-20T01:22:04ZengMDPI AGViruses1999-49152020-05-0112556810.3390/v12050568Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral InfectivityShuohui Liu0Rebecca Kaddis Maldonado1Tiffiny Rye-McCurdy2Christiana Binkley3Aissatou Bah4Eunice C. Chen5Breanna L. Rice6Leslie J. Parent7Karin Musier-Forsyth8Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USADepartments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USADepartment of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USADepartment of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USADepartment of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USADepartments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USADepartments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USADepartments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USADepartment of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USARetroviruses package their full-length, dimeric genomic RNA (gRNA) via specific interactions between the Gag polyprotein and a “Ψ” packaging signal located in the gRNA 5′-UTR. Rous sarcoma virus (RSV) gRNA has a contiguous, well-defined Ψ element, that directs the packaging of heterologous RNAs efficiently. The simplicity of RSV Ψ makes it an informative model to examine the mechanism of retroviral gRNA packaging, which is incompletely understood. Little is known about the structure of dimerization initiation sites or specific Gag interaction sites of RSV gRNA. Using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 5′-leader RNA for the first time. We identified a putative bipartite dimerization initiation signal (DIS), and mutation of both sites was required to significantly reduce dimerization in vitro. These mutations failed to reduce viral replication, suggesting that in vitro dimerization results do not strictly correlate with in vivo infectivity, possibly due to additional RNA interactions that maintain the dimers in cells. UV crosslinking-coupled SHAPE (XL-SHAPE) was next used to determine Gag-induced RNA conformational changes, revealing G218 as a critical Gag contact site. Overall, our results suggest that disruption of either of the DIS sequences does not reduce virus replication and reveal specific sites of Gag–RNA interactions.https://www.mdpi.com/1999-4915/12/5/568Rous sarcoma virus (RSV)retrovirusesGag polyproteinpackaging signal (Ψ)selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE)RNA secondary structure |
| spellingShingle | Shuohui Liu Rebecca Kaddis Maldonado Tiffiny Rye-McCurdy Christiana Binkley Aissatou Bah Eunice C. Chen Breanna L. Rice Leslie J. Parent Karin Musier-Forsyth Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity Viruses Rous sarcoma virus (RSV) retroviruses Gag polyprotein packaging signal (Ψ) selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) RNA secondary structure |
| title | Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity |
| title_full | Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity |
| title_fullStr | Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity |
| title_full_unstemmed | Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity |
| title_short | Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity |
| title_sort | rous sarcoma virus genomic rna dimerization capability in vitro is not a prerequisite for viral infectivity |
| topic | Rous sarcoma virus (RSV) retroviruses Gag polyprotein packaging signal (Ψ) selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) RNA secondary structure |
| url | https://www.mdpi.com/1999-4915/12/5/568 |
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