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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.

The subgroup A through E avian sarcoma and leukosis viruses (ASLV(A) through ASLV(E)) are a group of highly related alpharetroviruses that have evolved their envelope glycoproteins to use different receptors to enable efficient virus entry due to host resistance and/or to expand host range. Previously, we demonstrated that ASLV(A) in the presence of a competitor to the subgroup A Tva receptor, SUA-rIgG immunoadhesin, evolved to use other receptor options. The selected mutant virus, RCASBP(A)Δ155-160, modestly expanded its use of the Tvb and Tvc receptors and possibly other cell surface proteins while maintaining the binding affinity to Tva. In this study, we further evolved the Δ155-160 virus with the genetic selection pressure of a soluble form of the Tva receptor that should force the loss of Tva binding affinity in the presence of the Δ155-160 mutation. Viable ASLVs were selected that acquired additional mutations in the Δ155-160 Env hypervariable regions that significantly broadened receptor usage to include Tvb and Tvc as well as retaining the use of Tva as a receptor determined by receptor interference assays. A similar deletion in the hr1 hypervariable region of the subgroup C ASLV glycoproteins evolved to broaden receptor usage when selected on Tvc-negative cells.

The Czech scientist Jan Svoboda was a pioneer of Rous sarcoma virus (RSV). In the 1960s, before the discovery of reverse transcriptase, he demonstrated the long-term persistence of the viral genome in non-productive mammalian cells, and he supported the DNA provirus hypothesis of Howard Temin. He showed how the virus can be rescued in the infectious form and elucidated the replication-competent nature of the Prague strain of RSV later used for the identification of the src oncogene. His studies straddled molecular oncology and virology, and he remained an active contributor to the field until his death last year. Throughout the 50 years that I was privileged to know Svoboda as my mentor and friend, I admired his depth of scientific inquiry and his steadfast integrity in the face of political oppression.