Institute of Virology, Medical Faculty, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
Institute of Medical Microbiology and Hospital Hygiene, Medical Faculty, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
Int J Mol Sci. 2019 Mar 3;20(5):1088. doi: 10.3390/ijms20051088.
Transcription of the HIV-1 provirus generates a viral pre-mRNA, which is alternatively spliced into more than 50 HIV-1 mRNAs encoding all viral proteins. Regulation of viral alternative splice site usage includes the presence of splicing regulatory elements (SREs) which can dramatically impact RNA expression and HIV-1 replication when mutated. Recently, we were able to show that two viral SREs, G-2 and ESE, are important players in the generation of viral , and mRNAs. Furthermore, we demonstrated that masking these SREs by transfected locked nucleic acid (LNA) mixmers affect the viral splicing pattern and viral particle production. With regard to the development of future therapeutic LNA mixmer-based antiretroviral approaches, we delivered the G-2 and the ESE LNA mixmers "nakedly", without the use of transfection reagents (gymnosis) into HIV-1 infected cells. Surprisingly, we observed that gymnotically-delivered LNA mixmers accumulated in the cytoplasm, and seemed to co-localize with GW bodies and induced degradation of mRNAs containing their LNA target sequence. The G-2 and the ESE LNA-mediated RNA degradation resulted in abrogation of viral replication in HIV-1 infected Jurkat and PM1 cells as well as in PBMCs.
HIV-1 前病毒的转录生成病毒前 mRNA，该前 mRNA 经过可变剪接可生成编码所有病毒蛋白的 50 多种 HIV-1 mRNA。病毒可变剪接位点使用的调节包括存在剪接调节元件（SREs），当这些 SREs 发生突变时，可显著影响 RNA 表达和 HIV-1 复制。最近，我们能够证明两个病毒 SREs（G-2 和 ESE）是生成病毒 、 和 mRNA 的重要因素。此外，我们证明通过转染锁核酸（LNA）混合体来掩盖这些 SREs 会影响病毒剪接模式和病毒颗粒的产生。关于未来基于治疗性 LNA 混合体的抗逆转录病毒方法的开发，我们将 G-2 和 ESE 的 LNA 混合体“裸转”（无需使用转染试剂）到 HIV-1 感染的细胞中。令人惊讶的是，我们观察到，裸转的 LNA 混合体在细胞质中积累，并且似乎与 GW 体共定位，并诱导其 LNA 靶序列所在的 mRNA 降解。G-2 和 ESE 的 LNA 介导的 RNA 降解导致 HIV-1 感染的 Jurkat 和 PM1 细胞以及 PBMC 中的病毒复制被阻断。
