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Open AccessArticle

Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity

1
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
2
Departments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USA
3
Department of Microbiology & Immunology, Penn State College of Medicine, Hershey, PA 17033, USA
*
Author to whom correspondence should be addressed.
Viruses 2020, 12(5), 568; https://doi.org/10.3390/v12050568
Received: 27 April 2020 / Revised: 19 May 2020 / Accepted: 20 May 2020 / Published: 22 May 2020
(This article belongs to the Special Issue The 11th International Retroviral Nucleocapsid and Assembly Symposium)
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. View Full-Text
Keywords: Rous sarcoma virus (RSV); retroviruses; Gag polyprotein; packaging signal (Ψ); selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE); RNA secondary structure; RNA structure-probing; dimerization initiation signal (DIS); RNA–protein interactions; UV crosslinking-coupled SHAPE (XL-SHAPE) Rous sarcoma virus (RSV); retroviruses; Gag polyprotein; packaging signal (Ψ); selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE); RNA secondary structure; RNA structure-probing; dimerization initiation signal (DIS); RNA–protein interactions; UV crosslinking-coupled SHAPE (XL-SHAPE)
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Liu, S.; Kaddis Maldonado, R.; Rye-McCurdy, T.; Binkley, C.; Bah, A.; Chen, E.C.; Rice, B.L.; Parent, L.J.; Musier-Forsyth, K. Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity. Viruses 2020, 12, 568.

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