The Intracellular DNA Sensor IFI16 Gene Acts as Restriction Factor for Human Cytomegalovirus Replication

Human interferon (IFN)-inducible IFI16 protein, an innate immune sensor of intracellular DNA, modulates various cell functions, however, its role in regulating virus growth remains unresolved. Here, we adopt two approaches to investigate whether IFI16 exerts pro- and/or anti-viral actions. First, the IFI16 gene was silenced using specific small interfering RNAs (siRNA) in human embryo lung fibroblasts (HELF) and replication of DNA and RNA viruses evaluated. IFI16-knockdown resulted in enhanced replication of Herpesviruses, in particular, Human Cytomegalovirus (HCMV). Consistent with this, HELF transduction with a dominant negative form of IFI16 lacking the PYRIN domain (PYD) enhanced the replication of HCMV. Second, HCMV replication was compared between HELFs overexpressing either the IFI16 gene or the LacZ gene. IFI16 overexpression decreased both virus yield and viral DNA copy number. Early and late, but not immediate-early, mRNAs and proteins were strongly down-regulated, thus IFI16 may exert its antiviral effect by impairing viral DNA synthesis. Constructs with the luciferase reporter gene driven by deleted or site-specific mutated forms of the HCMV DNA polymerase (UL54) promoter demonstrated that the inverted repeat element 1 (IR-1), located between −54 and −43 relative to the transcription start site, is the target of IFI16 suppression. Indeed, electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated that suppression of the UL54 promoter is mediated by IFI16-induced blocking of Sp1-like factors. Consistent with these results, deletion of the putative Sp1 responsive element from the HCMV UL44 promoter also relieved IFI16 suppression. Together, these data implicate IFI16 as a novel restriction factor against HCMV replication and provide new insight into the physiological functions of the IFN-inducible gene IFI16 as a viral restriction factor.

人类干扰素（IFN）诱导的IFI16蛋白是一种胞内DNA天然免疫感受器，可调控多种细胞功能，但其在调控病毒增殖中的作用仍未明确。本研究采用两种方法探究IFI16是否兼具促病毒与抗病毒活性。首先，利用特异性小干扰RNA（siRNA）在人胚肺成纤维细胞（HELF）中沉默IFI16基因，并对DNA病毒与RNA病毒的复制水平进行评估。结果显示，IFI16敲低可增强疱疹病毒的复制能力，尤以人类巨细胞病毒（HCMV）为显著。与此一致，向HELF细胞转染缺失PYRIN结构域（PYD）的显性负突变型IFI16，同样可促进HCMV的复制。其次，分别比较过表达IFI16基因与LacZ基因的HELF细胞中HCMV的复制情况。实验结果表明，IFI16过表达可同时降低病毒滴度与病毒DNA拷贝数。病毒的早期、晚期mRNA与蛋白表达均显著下调，但即刻早期mRNA与蛋白无此变化，提示IFI16可能通过抑制病毒DNA合成发挥抗病毒效应。采用由缺失或位点特异性突变的HCMV DNA聚合酶（UL54）启动子驱动的荧光素酶报告基因构建体进行实验，结果证实，位于转录起始位点-54至-43区域的反向重复元件1（IR-1）是IFI16发挥抑制作用的靶点。进一步的电泳迁移率变动分析（EMSA）与染色质免疫沉淀（ChIP）实验证明，UL54启动子的抑制效应是通过IFI16阻断类Sp1转录因子实现的。与此结果一致，从HCMV UL44启动子中删除推定的Sp1应答元件后，也可解除IFI16的抑制作用。综上，本研究证实IFI16是一种新型抗HCMV复制的宿主限制因子，并为IFN诱导基因IFI16作为病毒限制因子的生理功能提供了新的研究视角。