Structural remodeling of the HIV-1 genomic RNA by EMCV IRES insertion impairs viral maturation and fitness

The HIV-1 genomic RNA contains highly structured cis-acting elements that regulate multiple stages of the viral replication cycle, including translation, genome packaging, and particle maturation. Disruption of these RNA structural networks may therefore compromise viral fitness and infectivity. To investigate the impact of large-scale RNA structural remodeling on HIV-1 replication, we analyzed an HIV-1 infectious clone containing the EMCV Internal Ribosome Entry Site (IRES) followed by the gfp reporter gene inserted downstream of nef (NL4-3-IRES). In silico RNA folding analyses predicted extensive global and local conformational changes throughout the viral genomic RNA induced by EMCV IRES insertion. Functionally, NL4-3-IRES exhibited a ~2.5-fold reduction in viral infectivity associated with impaired PR-mediated Gag processing and reduced viral maturation. IRES-containing viral particles displayed decreased incorporation of mature p24-Capsid, Reverse Transcriptase, and Integrase proteins, consistent with defects in viral particle composition. Polysome profiling further revealed altered ribosome association patterns among distinct HIV-1 RNA species. Importantly, similar replication defects were also observed in an independent HIV-1 background. Together, these findings demonstrate that structural remodeling of the HIV-1 genomic RNA induced by EMCV IRES insertion disrupts viral maturation and reduces HIV-1 infectivity, reinforcing the critical role of RNA structural organization in the HIV-1 replication cycle.人类免疫缺陷病毒1型（HIV-1）的基因组RNA携带有高度结构化的顺式作用元件（cis-acting elements），可调控病毒复制周期（viral replication cycle）的多个阶段，包括翻译、基因组包装（genome packaging）与病毒颗粒成熟（particle maturation）。因此，这些RNA结构网络的破坏可能会损害病毒的复制适应性（viral fitness）与感染性（infectivity）。为探究大规模RNA结构重塑（large-scale RNA structural remodeling）对HIV-1复制的影响，本研究分析了一款携带脑心肌炎病毒（Encephalomyocarditis Virus, EMCV）内部核糖体进入位点（Internal Ribosome Entry Site, IRES）、且在nef基因下游插入绿色荧光蛋白（green fluorescent protein, GFP）报告基因（reporter gene）的HIV-1感染性克隆（infectious clone，NL4-3-IRES）。 虚拟RNA折叠分析（in silico RNA folding analyses）预测，EMCV IRES的插入会诱导HIV-1基因组RNA发生广泛的全局与局部构象变化（conformational changes）。功能实验结果显示，NL4-3-IRES的病毒感染性（viral infectivity）下降约2.5倍，该现象与蛋白酶（Protease, PR）介导的Gag蛋白加工（Gag processing）受损以及病毒成熟能力降低密切相关。携带IRES的病毒颗粒（viral particles）中，成熟p24衣壳蛋白（p24-Capsid）、逆转录酶（Reverse Transcriptase）与整合酶（Integrase）的整合量均显著下降，这与病毒颗粒组成存在缺陷的表型一致。多聚核糖体谱分析（polysome profiling）进一步揭示，不同HIV-1 RNA物种（RNA species）的核糖体结合模式（ribosome association patterns）发生了改变。值得注意的是，在另一独立的HIV-1遗传背景（background）中也观察到了类似的复制缺陷。 综上，本研究结果表明，EMCV IRES插入诱导的HIV-1基因组RNA结构重塑会破坏病毒成熟过程并降低HIV-1感染性，进一步证实了RNA结构组织在HIV-1复制周期中的关键作用。