Impact of IRES sequence insertrion in the HIV-1 genome

The HIV-1 genome contains several RNA structures involved with viral genomic RNA packaging; which is directly related with viral maturation . Thus, mutations leading to structural alterations in the viral genomic RNA have the potential to impact these processes and consequently impair viral infectivity. The insertion of a highly structured RNA element at the 3’-end of the HIV-1 genome may cause deleterious alterations or hide signalling sequences important for viral replication. To characterise this further, we took advantage of an HIV-1 infectious clone harbouring a ECMV Internal Ribosome Entry Site (IRES) sequence followed by the gfp reporter gene introduced downstream of the nef gene (NL4-3-IRES). In cells, NL4-3-IRES was 2.5-fold less infectious than the wild type NL4-3 and had a 2-fold reduction in Gag processing. Mature particles showed less incorporation of p24-Capsid, Reverse Transcriptase, and Integrase proteins within NL4-3-IRES viral particles. In silico molecular modeling analysis revealed that the genomic RNA of NL4-3-IRES had several conformational changes compared with the wild type NL4-3 sequence, especially in regions related to viral maturation, demonstrating that the presence of an IRES element within the 3’ end of the HIV-1 genome has profound impacts on virus assembly and maturation and consequently reduced viral infectivity. This dataset consists in Western blot, RT-qPCR, ELISA and infectivity assay results. Also, results from a iodixanol gradiente, in which each fraction was measured by optical density, can be found. The data can be interpreted by comparing the values of the measuring units from each assay or experiment. The secondary structure of the genome of wild type HIV-1, depleted of not fo Nef gene and with or without IRES sequence, was solved. It can be interpreted by comparing the differences, both local and global, in their structures.HIV-1基因组包含多种参与病毒基因组RNA包装的RNA结构，该过程与病毒成熟直接相关。因此，导致病毒基因组RNA结构改变的突变，有可能影响上述过程，进而削弱病毒感染性。在HIV-1基因组3'端插入高度结构化的RNA元件，可能引发有害的结构改变，或掩盖病毒复制所需的关键信号序列。为进一步解析这一现象，本研究利用一株携带脑心肌炎病毒（Encephalomyocarditis virus, ECMV）内部核糖体进入位点（Internal Ribosome Entry Site, IRES）序列，且在nef基因下游插入绿色荧光蛋白（green fluorescent protein, GFP）报告基因的HIV-1感染性克隆（命名为NL4-3-IRES）开展实验。细胞实验结果显示，NL4-3-IRES的感染性较野生型NL4-3低2.5倍，且Gag蛋白加工效率降低2倍。成熟病毒粒子中，NL4-3-IRES颗粒内的p24衣壳蛋白、逆转录酶（Reverse Transcriptase, RT）及整合酶（Integrase, IN）的掺入量均有所减少。计算机分子建模分析表明，与野生型NL4-3序列相比，NL4-3-IRES的基因组RNA存在多处构象变化，尤其在与病毒成熟相关的区域，这证实了HIV-1基因组3'端插入IRES元件会对病毒组装与成熟产生显著影响，进而降低病毒感染性。本数据集包含蛋白质印迹（Western blot）、实时定量逆转录聚合酶链反应（RT-qPCR）、酶联免疫吸附测定（ELISA）及感染性测定的相关结果。此外，本数据集还包含碘克沙醇梯度离心实验结果，该实验中各梯度组分均通过光密度进行了检测。研究者可通过比较各测定或实验的测量单位数值对数据进行解读。本研究还解析了野生型HIV-1基因组、缺失nef基因的HIV-1基因组，以及带有或不带IRES序列的HIV-1基因组的二级结构，可通过比较其局部与全局的结构差异对数据进行解读。