Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.
Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.
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DOI：
10.1128/mbio.01553-16
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发表时间：
2016-11-15
期刊：
影响因子：
6.4
通讯作者：
Cristea IM
中科院分区：
文献类型：
--
作者：
Diner BA;Lum KK;Toettcher JE;Cristea IM
人干扰素诱导蛋白IFI16是一种重要的抗病毒因子，其结合核病毒DNA并促进抗病毒应答。在这里，我们定义了IFI16在空间和时间上的动态及其与DNA传感器环二核苷酸GMP-AMP合酶（cGAS）的不同功能。活细胞成像揭示了多相的IFI16再分布，首先是在核周围的病毒进入位点，然后是单纯疱疹病毒1型（HSV-1）和人巨细胞病毒（HCMV）感染后的核质点。光遗传学和活细胞显微镜建立IFI16 pyrin结构域所需的核周边定位和寡聚化。此外，使用蛋白质组学，我们定义了IFI16 pyrin和HIN200结构域的签名蛋白质相互作用，并证明了pyrin对于IFI16与抗病毒蛋白PML和cGAS相互作用的必要性。我们在原代成纤维细胞中使用成簇规则间隔短回文重复序列（CRISPR）/Cas9介导的敲除来探测IFI16、cGAS和PML参与的信号传导途径。虽然IFI16诱导细胞因子，但只有cGAS激活STING/TBK-1/IRF 3和HSV-1和HCMV感染后的凋亡反应。DNA刺激后的cGAS依赖性细胞凋亡需要环状二核苷酸和STING的酶促产生。我们发现，IFI16，而不是cGAS或PML，抑制HSV-1基因表达，降低病毒滴度。这表明病毒基因表达的调节可能比抗病毒细胞因子的诱导对病毒复制具有更大的屏障作用。总之，我们的研究结果建立了IFI16和cGAS针对疱疹病毒的协调和独特的抗病毒功能。哺乳动物细胞如何检测和响应在细胞核中复制的DNA病毒还知之甚少。在这里，我们破译了两种病毒DNA传感器，IFI16和cGAS，在感染两种疱疹病毒，单纯疱疹病毒1（HSV-1）和人巨细胞病毒（HCMV）后的主动免疫信号传导过程中的不同功能。我们发现，IFI16在进入疱疹病毒基因组的核周围快速寡聚化，从而在转录上抑制病毒基因表达并限制病毒复制能力。我们进一步证明，IFI16不启动经典STING/TBK-1/IRF3信号传导途径的上游激活，而是下游抗病毒细胞因子表达所需的。相比之下，我们发现，在疱疹病毒感染期间的DNA传感，cGAS触发细胞凋亡的STING依赖性的方式。我们的活细胞成像、基于质谱的蛋白质组学、基于CRISPR的细胞测定和光遗传学强调了综合方法在揭示针对病原体的复杂细胞反应方面的价值。
The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16 rapidly oligomerizes at incoming herpesvirus genomes at the nuclear periphery to transcriptionally repress viral gene expression and limit viral replicative capacity. We further demonstrate that IFI16 does not initiate upstream activation of the canonical STING/TBK-1/IRF3 signaling pathway but is required for downstream antiviral cytokine expression. In contrast, we find that, upon DNA sensing during herpesvirus infection, cGAS triggers apoptosis in a STING-dependent manner. Our live-cell imaging, mass spectrometry-based proteomics, CRISPR-based cellular assays, and optogenetics underscore the value of integrative approaches to uncover complex cellular responses against pathogens.