Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability
Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability
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DOI：
10.1128/jvi.00923-16
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发表时间：
2016-09-01
影响因子：
5.4
通讯作者：
Landolfo, Santo
中科院分区：
文献类型：
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作者：
Biolatti, Matteo;Dell'Oste, Valentina;Landolfo, Santo
干扰素-γ-诱导蛋白16(IFI16)在人巨细胞病毒(HCMV)的内在抵抗中起关键作用，它在感染后的最初几个小时作为病毒DNA感受器，在感染的后期作为病毒基因转录的抑制因子。以往对HCMV复制的研究表明，IFI16与病毒蛋白激酶pUL97结合，经历磷酸化，并重新定位到感染细胞的细胞质中。在这项研究中，我们证明了被膜蛋白pp65(PUL83)将IFI16招募到UL54基因的启动子上，并下调病毒复制，如使用缺乏pp65表达的HCMV突变体v65Stop所显示的那样。有趣的是，在HCMV感染的后期，IFI16通过与pp65的相互作用而稳定下来，这与在单纯疱疹病毒1型(HSV-1)感染的细胞中观察到的IFI16的降解相反。此外，我们还发现，除了pUL97外，它的细胞质转运严格依赖于pp65，就像HCMV突变体RV-VM1所证明的那样，它表达一种无法转运到细胞质的pp65。因此，这些数据揭示了pp65的双重作用：在早期感染期间，它调节即刻早期和早期基因启动子上的IFI16活性；随后，它将IFI16从细胞核离域到细胞质，从而稳定和保护它免受降解。总之，这些数据证实了pp65/IFI16相互作用体的一个新的活性，该相互作用体参与调节HCMV复制的早期和晚期UL54基因的表达和IFI16的稳定性。使用一种缺乏被膜蛋白pp65(V65Stop)的突变病毒，我们证明了pp65将IFI16招募到早期UL54基因启动子。作为对其限制活性的假定中和，pp65支持IFI16的核质输出，这在病毒突变体RV-VM1表达核保留pp65的情况下得到了证实。这些数据揭示了pp65在IFI16调控中的双重作用：在HCMV感染的早期，它有助于病毒逃避IFI16的限制活性，而在以后的时间点，它促进IFI16的核离域，从而稳定和保护它免受降解。在目前的工作中，我们进一步阐明了HCMV依赖于克服细胞内天然免疫限制的机制，并为DNA传感限制因子IFI16在HCMV感染过程中的相关性提供了新的见解。
A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-gamma-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication.IMPORTANCEThe DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the viral mutant RV-VM1 expressing a nuclearly retained pp65. These data reveal a dual role of pp65 in IFI16 regulation: in the early phase of HCMV infection, it contributes to viral evasion from IFI16 restriction activity, while at later time points, it promotes the nuclear delocalization of IFI16, thereby stabilizing and protecting it from degradation. In the present work, we further clarify the mechanisms HCMV relies on to overcome intracellular innate immune restriction and provide new insights into the relevance of DNA-sensing restriction factor IFI16 during HCMV infection.