Gene Therapy Center University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.
Retrovirology. 2011 Jun 24;8:51. doi: 10.1186/1742-4690-8-51.
The process of HIV-1 genomic RNA (gRNA) encapsidation is governed by a number of viral encoded components, most notably the Gag protein and gRNA cis elements in the canonical packaging signal (ψ). Also implicated in encapsidation are cis determinants in the R, U5, and PBS (primer binding site) from the 5' untranslated region (UTR). Although conventionally associated with nuclear export of HIV-1 RNA, there is a burgeoning role for the Rev/RRE in the encapsidation process. Pleiotropic effects exhibited by these cis and trans viral components may confound the ability to examine their independent, and combined, impact on encapsidation of RNA into HIV-1 viral particles in their innate viral context. We systematically reconstructed the HIV-1 packaging system in the context of a heterologous murine leukemia virus (MLV) vector RNA to elucidate a mechanism in which the Rev/RRE system is central to achieving efficient and specific encapsidation into HIV-1 viral particles.
We show for the first time that the Rev/RRE system can augment RNA encapsidation independent of all cis elements from the 5' UTR (R, U5, PBS, and ψ). Incorporation of all the 5' UTR cis elements did not enhance RNA encapsidation in the absence of the Rev/RRE system. In fact, we demonstrate that the Rev/RRE system is required for specific and efficient encapsidation commonly associated with the canonical packaging signal. The mechanism of Rev/RRE-mediated encapsidation is not a general phenomenon, since the combination of the Rev/RRE system and 5' UTR cis elements did not enhance encapsidation into MLV-derived viral particles. Lastly, we show that heterologous MLV RNAs conform to transduction properties commonly associated with HIV-1 viral particles, including in vivo transduction of non-dividing cells (i.e. mouse neurons); however, the cDNA forms are episomes predominantly in the 1-LTR circle form.
Premised on encapsidation of a heterologous RNA into HIV-1 viral particles, our findings define a functional HIV-1 packaging system as comprising the 5' UTR cis elements, Gag, and the Rev/RRE system, in which the Rev/RRE system is required to make the RNA amenable to the ensuing interaction between Gag and the canonical packaging signal for subsequent encapsidation.
HIV-1 基因组 RNA（gRNA）的包装过程受到多种病毒编码成分的控制，其中最重要的是 Gag 蛋白和规范包装信号（ψ）中的 gRNA 顺式元件。5'非翻译区（UTR）中的 R、U5 和 PBS（引物结合位点）中的顺式决定因素也与包装有关。虽然 HIV-1 RNA 的核输出通常与 Rev/RRE 相关，但 Rev/RRE 在包装过程中也发挥着越来越重要的作用。这些顺式和反式病毒成分表现出的多效性可能会影响到检查它们对 HIV-1 病毒颗粒中 RNA 包装的独立和综合影响的能力，而这种包装是在其固有病毒环境中进行的。我们系统地在异源鼠白血病病毒（MLV）载体 RNA 的背景下重建了 HIV-1 包装系统，以阐明 Rev/RRE 系统在实现高效和特异性将 RNA 包装到 HIV-1 病毒颗粒中的核心机制。
我们首次表明，Rev/RRE 系统可以在没有 5'UTR（R、U5、PBS 和 ψ）中所有顺式元件的情况下增强 RNA 包装。在没有 Rev/RRE 系统的情况下，包含所有 5'UTR 顺式元件并不能增强 RNA 包装。事实上，我们证明 Rev/RRE 系统是实现与规范包装信号相关的特异性和高效包装所必需的。Rev/RRE 介导的包装机制不是一个普遍现象，因为 Rev/RRE 系统和 5'UTR 顺式元件的组合并不能增强 MLV 衍生病毒颗粒的包装。最后，我们表明，异源 MLV RNAs 符合与 HIV-1 病毒颗粒相关的转导特性，包括非分裂细胞（即小鼠神经元）的体内转导；然而，cDNA 形式主要以 1-LTR 环形式存在于染色体外。
基于将异源 RNA 包装到 HIV-1 病毒颗粒中，我们的发现定义了一个功能性的 HIV-1 包装系统，该系统包含 5'UTR 顺式元件、Gag 和 Rev/RRE 系统，其中 Rev/RRE 系统是使 RNA 能够与随后的 Gag 与规范包装信号之间的相互作用相容的必需条件，以进行随后的包装。
