将基因组 RNA (gRNA) 包装到逆转录病毒颗粒中依赖于包装信号 (Psi) 的 Gag 前体的特异性识别，该信号通过长程相互作用 (LRI) 维持复杂的二级结构。然而，目前尚不清楚 Gag 与 Psi 的单独结合是否足以促进 RNA 包装以及 LRI 在此过程中发挥什么作用。使用小鼠乳腺肿瘤病毒 (MMTV)，我们研究了 4 种 LRI 中的突变对 gRNA 结构和功能的影响。我们的研究结果揭示了存在未被怀疑的扩展 LRI，而 hSHAPE 表明维持类似野生型的 Psi 结构对于有效包装至关重要。令人惊讶的是，过滤器结合测定表明，大多数突变体，无论其包装能力如何，都表现出与 Pr77Gag 的显着结合，这表明 Gag 与 Psi 的结合不足以实现有效包装。足迹实验表明，当 Pr77Gag 与 Psi 内的 2 个特定位点结合时，会促进有效的 RNA 包装，而与 Psi 中其他位置的结合不会导致有效的包装。总而言之，我们的结果表明，Psi/Pr77Gag 复合物的 3D 结构调节 gRNA 周围病毒颗粒的组装，从而能够有效区分也可能有效结合 Gag 的其他病毒和细胞 RNA。版权所有：© 2024 Prabhu 等人。这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原始作者和来源。

The packaging of genomic RNA (gRNA) into retroviral particles relies on the specific recognition by the Gag precursor of packaging signals (Psi), which maintain a complex secondary structure through long-range interactions (LRIs). However, it remains unclear whether the binding of Gag to Psi alone is enough to promote RNA packaging and what role LRIs play in this process. Using mouse mammary tumor virus (MMTV), we investigated the effects of mutations in 4 proposed LRIs on gRNA structure and function. Our findings revealed the presence of an unsuspected extended LRI, and hSHAPE revealed that maintaining a wild-type-like Psi structure is crucial for efficient packaging. Surprisingly, filter-binding assays demonstrated that most mutants, regardless of their packaging capability, exhibited significant binding to Pr77Gag, suggesting that Gag binding to Psi is insufficient for efficient packaging. Footprinting experiments indicated that efficient RNA packaging is promoted when Pr77Gag binds to 2 specific sites within Psi, whereas binding elsewhere in Psi does not lead to efficient packaging. Taken together, our results suggest that the 3D structure of the Psi/Pr77Gag complex regulates the assembly of viral particles around gRNA, enabling effective discrimination against other viral and cellular RNAs that may also bind Gag efficiently.Copyright: © 2024 Prabhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.