Guney Mehmet Hakan, Nagalekshmi Karthika, McCauley Sean Matthew, Carbone Claudia, Aydemir Ozkan, Luban Jeremy
Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
These authors contributed equally.
bioRxiv. 2023 Dec 12:2023.11.17.567619. doi: 10.1101/2023.11.17.567619.
Antiretroviral therapy (ART) suppresses HIV-1 viremia and prevents progression to AIDS. Nonetheless, chronic inflammation is a common problem for people living with HIV-1 on ART. One possible cause of inflammation is ongoing transcription from HIV-1 proviruses, whether or not the sequences are competent for replication. Previous work has shown that intron-containing RNA expressed from the HIV-1 provirus in primary human blood cells, including CD4 T cells, macrophages, and dendritic cells, activates type 1 interferon. This activation required HIV-1 and was blocked by the XPO1 (CRM1)-inhibitor leptomycin. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with shRNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the IFN-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with non-targetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant inhibitory CARD-deletion or phosphomimetic point mutations, indicates that IFIH1 filament formation, dephosphorylation, and association with MAVS, are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1-knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 was revealed by formaldehyde crosslinking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.
抗逆转录病毒疗法（ART）可抑制HIV-1病毒血症并预防疾病进展至艾滋病。尽管如此，慢性炎症仍是接受ART治疗的HIV-1感染者的常见问题。炎症的一个可能原因是HIV-1前病毒的持续转录，无论其序列是否具有复制能力。先前的研究表明，在包括CD4 T细胞、巨噬细胞和树突状细胞在内的原代人血细胞中，由HIV-1前病毒表达的含内含子RNA可激活1型干扰素。这种激活需要HIV-1参与，并被XPO1（CRM1）抑制剂来普霉素阻断。为了确定检测HIV-1前病毒表达的含内含子RNA所需的天然免疫受体，在人单核细胞衍生的树突状细胞中，使用靶向21个候选基因的表达shRNA的慢病毒载体进行了功能缺失筛选。在测试的候选基因中，只有敲低XPO1（CRM1）、IFIH1（MDA5）或MAVS才能阻止干扰素刺激基因ISG15的激活。通过用不可靶向的IFIH1编码序列挽救敲低，证明了IFIH1蛋白的重要性。尼帕病毒V蛋白以及IFIH1显性抑制性CARD缺失或磷酸模拟点突变对HIV-1诱导的ISG15的抑制作用表明，IFIH1丝的形成、去磷酸化以及与MAVS的结合，都是响应HIV-1转导的天然免疫激活所必需的。由于IFIH1和DDX58（RIG-I）均通过MAVS发出信号，DDX58敲低对激活无影响这一事实证明了IFIH1对HIV-1 RNA检测的特异性。RNA测序表明，树突状细胞中IFIH1的敲低全面破坏了干扰素刺激基因的诱导。最后，通过甲醛交联免疫沉淀（f-CLIP）揭示了IFIH1对未剪接HIV-1 RNA的特异性富集。这些结果表明，IFIH1是HIV-1前病毒含内含子RNA激活天然免疫所必需的，并且可能导致HIV-1感染者的慢性炎症。
