Rous Sarcoma Virus Genomic RNA Dimerization Capability In Vitro Is Not a Prerequisite for Viral Infectivity
Shuohui Liu,
Rebecca Kaddis Maldonado,
Tiffiny Rye-McCurdy,
Christiana Binkley,
Aissatou Bah,
Eunice C. Chen,
Breanna L. Rice,
Leslie J. Parent,
Karin Musier-Forsyth
Affiliations
Shuohui Liu
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Rebecca Kaddis Maldonado
Departments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USA
Tiffiny Rye-McCurdy
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Christiana Binkley
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Aissatou Bah
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
Eunice C. Chen
Departments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USA
Breanna L. Rice
Departments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USA
Leslie J. Parent
Departments of Medicine, Division of Infectious Diseases and Epidemiology, Penn State College of Medicine, Hershey, PA 17033, USA
Karin Musier-Forsyth
Department of Chemistry and Biochemistry, Center for Retroviral Research, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
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.
