Hughes Ivy K, Kharytonchyk Siarhei, Ramaswamy Sita, Jalloh Sallieu, Hood James B, He Xianbao, Henderson Andrew J, Akiyama Hisashi, Telesnitsky Alice, Gummuluru Suryaram
bioRxiv. 2025 Aug 25:2025.08.20.671346. doi: 10.1101/2025.08.20.671346.
Heterogenous transcription start site (TSS) usage dictates the structure and function of unspliced HIV-1 RNAs (usRNA). We and others have previously reported that expression and Rev/CRM1-mediated nuclear export of HIV-1 usRNA in macrophages activates MDA5, MAVS, and innate immune signaling cascades. In this study, we reveal that MDA5 sensing of viral usRNA is strictly determined by TSS, 5' leader structure, and RNA function. We show that cap-sequestered HIV-1 usRNAs ( 1G) destined for viral genome packaging are specifically targeted by MDA5, while translation-destined ( 3G) usRNAs are remarkably immunologically silent. Using mutant viruses which express usRNA with altered 5' cap-exposed leader structure, or inclusion of a retroviral constitutive transport element which drives mRNA-like NXF1-dependent nuclear export of viral usRNA, we show that cap exposure and nuclear export pathway choice are major determinants of both lentiviral RNA immunogenicity and function. In total, we identify innate immune system evasion as a possible rationale for the universal conservation of heterogenous TSS usage among ancestral and extant HIV-1 isolates and shed light on how MDA5 fundamentally discriminates between self and non-self RNAs.
Innate immune activation is critical to both the process of initial infection establishment and ongoing chronic inflammation in HIV-1 infection. While MDA5 has been identified as the sensor which detects unspliced HIV-1 RNA produced in infected cells, it remains unclear how HIV-1 unspliced RNAs, which are generated by cellular transcriptional processes, are recognized as non-self. Here, we reveal that HIV-1 RNA function determines MDA5-driven immunogenicity. We show that only unspliced RNAs which traffic to membrane-associated viral assembly sites are immunogenic, while unspliced RNAs which are ribosomally translated to produce viral proteins are immunologically silent. These findings not only advance our knowledge of how the human innate immune system recognizes HIV-1 unspliced RNAs as foreign but also provide a rationale for the selective advantage to generate two pools of unspliced RNAs during HIV-1 replication.
异源转录起始位点（TSS）的使用决定了未剪接的HIV-1 RNA（usRNA）的结构和功能。我们和其他人之前曾报道，巨噬细胞中HIV-1 usRNA的表达以及Rev/CRM1介导的核输出会激活MDA5、MAVS和先天性免疫信号级联反应。在本研究中，我们揭示MDA5对病毒usRNA的识别严格由TSS、5'前导序列结构和RNA功能决定。我们表明，用于病毒基因组包装的帽封端HIV-1 usRNAs（1G）被MDA5特异性靶向，而用于翻译的（3G）usRNAs在免疫上显著沉默。使用表达具有改变的5'帽暴露前导序列结构的usRNA的突变病毒，或包含驱动病毒usRNA进行类似mRNA的NXF1依赖性核输出的逆转录病毒组成型转运元件，我们表明帽暴露和核输出途径的选择是慢病毒RNA免疫原性和功能的主要决定因素。总体而言，我们确定先天性免疫系统逃避是祖先和现存HIV-1分离株中异源TSS使用普遍保守的一个可能原因，并阐明了MDA5如何从根本上区分自身和非自身RNA。
先天性免疫激活对于HIV-1感染中初始感染建立过程和持续慢性炎症都至关重要。虽然MDA5已被确定为检测感染细胞中产生的未剪接HIV-1 RNA的传感器，但尚不清楚通过细胞转录过程产生的HIV-1未剪接RNA如何被识别为非自身。在此，我们揭示HIV-1 RNA功能决定了MDA5驱动的免疫原性。我们表明，只有转运到膜相关病毒组装位点的未剪接RNA具有免疫原性，而通过核糖体翻译产生病毒蛋白的未剪接RNA在免疫上是沉默的。这些发现不仅推进了我们对人类先天性免疫系统如何将HIV-1未剪接RNA识别为外来物的认识，还为HIV-1复制过程中产生两类未剪接RNA的选择优势提供了一个理由。
