The retroviral Gag precursor plays a central role in the selection and packaging of viral genomic RNA (gRNA) by binding to virus-specific packaging signal(s) (psi or ψ). Previously, we mapped the feline immunodeficiency virus (FIV) ψ to two discontinuous regions within the 5′ end of the gRNA that assumes a higher order structure harboring several structural motifs. To better define the region and structural elements important for gRNA packaging, we methodically investigated these FIV ψ sequences using genetic, biochemical, and structure–function relationship approaches. Our mutational analysis revealed that the unpaired U⁸⁵CUG⁸⁸ stretch within FIV ψ is crucial for gRNA encapsidation into nascent virions. High-throughput selective 2′ hydroxyl acylation analyzed by primer extension (hSHAPE) performed on wild type (WT) and mutant FIV ψ sequences, with substitutions in the U⁸⁵CUG⁸⁸ stretch, revealed that these mutations had limited structural impact and maintained nucleotides 80–92 unpaired, as in the WT structure. Since these mutations dramatically affected packaging, our data suggest that the single-stranded U⁸⁵CUG⁸⁸ sequence is important during FIV RNA packaging. Filter-binding assays performed using purified FIV Pr50^Gag on WT and mutant U⁸⁵CUG⁸⁸ ψ RNAs led to reduced levels of Pr50^Gag binding to mutant U⁸⁵CUG⁸⁸ ψ RNAs, indicating that the U⁸⁵CUG⁸⁸ stretch is crucial for ψ RNA–Pr50^Gag interactions. Delineating sequences important for FIV gRNA encapsidation should enhance our understanding of both gRNA packaging and virion assembly, making them potential targets for novel retroviral therapeutic interventions, as well as the development of FIV-based vectors for human gene therapy.

中文翻译：

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鉴定对猫免疫缺陷病毒基因组 RNA 包装至关重要的假定 Gag 结合位点

逆转录病毒 Gag 前体通过与病毒特异性包装信号（psi 或 ψ）结合，在病毒基因组 RNA (gRNA) 的选择和包装中发挥核心作用。此前，我们将猫免疫缺陷病毒 (FIV) ψ 映射到 gRNA 5' 端内的两个不连续区域，该区域呈现出包含多个结构基序的更高阶结构。为了更好地定义对 gRNA 包装重要的区域和结构元件，我们使用遗传、生化和结构-功能关系方法系统地研究了这些 FIV ψ 序列。我们的突变分析显示，未配对的 U⁸⁵CUG⁸⁸ 在 FIV 内延伸ψ 对于 gRNA 衣壳化为新生病毒粒子至关重要。通过对野生型 (WT) 和突变型 FIV ψ 序列进行引物延伸 (hSHAPE) 分析高通量选择性 2' 羟基酰化，并在 U⁸⁵⁸⁸ 拉伸表明，这些突变的结构影响有限，并保持 80-92 个核苷酸不配对，如 WT 结构中一样。由于这些突变极大地影响了包装，我们的数据表明单链 U⁸⁵CUG⁸⁸ 序列在 FIV RNA 包装过程中非常重要。使用纯化的 FIV Pr50^Gag 在 WT 和突变体 U⁸⁵⁸⁸ ψ RNA 导致 Pr50^Gag 结合水平降低突变体 U⁸⁵CUG⁸⁸ ψ RNA，表明 U< /span>互动。描绘对 FIV gRNA 衣壳化重要的序列应该会增强我们对 gRNA 包装和病毒粒子组装的理解，使它们成为新型逆转录病毒治疗干预措施以及开发基于 FIV 的人类基因治疗载体的潜在目标。^搞笑 拉伸对于 ψ RNA–Pr50 至关重要⁸⁸CUG⁸⁵