Department of Microbiology & Immunology, Stanford University SOM, Stanford, CA 94305, USA.
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru 560064, Karnataka, India.
Viruses. 2024 May 3;16(5):725. doi: 10.3390/v16050725.
The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.
登革热病毒是一种单链、正链 RNA 病毒，感染全球约 4 亿人。目前尚无已批准的抗病毒药物。基于 CRISPR 的筛选方法极大地加速了宿主因子的发现，这些因子对 DENV 感染至关重要，并且可以作为宿主定向抗病毒干预的靶点。在本研究中，我们进行了一项集中的 CRISPR（成簇规律间隔短回文重复）文库筛选，以发现对人类 Huh7 细胞中 DENV 感染至关重要的关键宿主因子，并确定蛋白激活干扰素诱导蛋白激酶（PACT）为 DENV 的新型促病毒因子。PACT 是一种双链 RNA 结合蛋白，通常在病毒感染细胞中激活抗病毒反应并阻断病毒复制。然而，在我们的研究中，我们观察到 PACT 在 DENV 感染中发挥促病毒作用，并特异性促进病毒 RNA 复制。PACT 的敲除导致感染细胞中 DENV RNA 和蛋白丰度显著下降，而外源性表达全长 PACT 则可挽救这一现象。对全局基因表达变化的分析表明，与感染野生型细胞相比，感染 PACT 敲除细胞中几种 ER 相关的促病毒基因（如 ERN1、DDIT3、HERPUD1 和 EIF2AK3）并未上调。因此，我们的研究表明 PACT 在促进 DENV 复制方面发挥了新的作用，可能通过调节 ER 相关促病毒基因的表达。
