MMTV RNA包装依赖于延伸的长程相互作用以促进Gag蛋白与包装信号的有效结合

MMTV RNA packaging requires an extended long-range interaction for productive Gag binding to packaging signals

PLOS BIOLOGY

影响因子:7.2

分区:生物学1区 Top / 生化与分子生物学1区生物学1区

发表日期:2024 Oct

作者: Suresha G Prabhu, Vineeta N Pillai, Lizna Mohamed Ali, Valérie Vivet-Boudou, Akhil Chameettachal, Serena Bernacchi, Farah Mustafa, Roland Marquet, Tahir A Rizvi

DOI: 10.1371/journal.pbio.3002827

摘要

基因组RNA（gRNA）包装到逆转录病毒颗粒中依赖Gag前体对包装信号（Psi）的特异性识别，该信号通过长程相互作用（LRIs）维持复杂的二级结构。然而，Gag仅与Psi结合是否足以促进RNA包装，以及LRIs在此过程中的作用尚不清楚。我们以鼠乳腺肿瘤病毒（MMTV）为模型，研究了4个假定LRIs突变对gRNA结构和功能的影响。研究发现存在一个未曾预料的延伸LRI，且hSHAPE分析显示维持类似野生型Psi结构对高效包装至关重要。令人惊讶的是，滤膜结合实验表明，大部分突变体，无论其包装能力如何，都能显著结合Pr77Gag，提示Gag与Psi的结合不足以实现高效包装。脚印实验显示，Pr77Gag在Psi的两个特定位置结合可促进RNA的高效包装，而在Psi其他位置的结合则不产生有效包装。综上所述，Psi/Pr77Gag复合物的三维结构调控病毒颗粒围绕gRNA的组装，从而实现对其他病毒及细胞RNA的有效识别和区分。

Abstract

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.