TY - JOUR
T1 - Rous sarcoma virus genomic RNA dimerization capability in vitro is not a prerequisite for viral infectivity
AU - Liu, Shuohui
AU - Maldonado, Rebecca Kaddis
AU - Rye-McCurdy, Tiffiny
AU - Binkley, Christiana
AU - Bah, Aissatou
AU - Chen, Eunice C.
AU - Rice, Breanna L.
AU - Parent, Leslie J.
AU - Musier-Forsyth, Karin
N1 - Funding Information:
Funding: This research was funded by NIH grants RO1 AI153216 (K.M.-F.), R01 CA76534 (L.J.P.), and F30 CA214010 (ECC).
Funding Information:
This research was funded by NIH grants RO1 AI153216 (K.M.-F.), R01 CA76534 (L.J.P.), and F30 CA214010 (ECC).
Publisher Copyright:
© 2020 by the authors.
PY - 2020/5
Y1 - 2020/5
N2 - 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 50-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 20-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 50-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.
AB - 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 50-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 20-hydroxyl acylation analyzed by primer extension (SHAPE), we probed the secondary structure of the entire RSV 50-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.
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U2 - 10.3390/v12050568
DO - 10.3390/v12050568
M3 - Article
C2 - 32455905
AN - SCOPUS:85085538825
SN - 1999-4915
VL - 12
JO - Viruses
JF - Viruses
IS - 5
M1 - 568
ER -