Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

Abstract

A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-␥-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. Human cytomegalovirus (HCMV) is a member of the Betaherpesvirinae subfamily of Herpesviridae. It is a widespread pathogen that infects the majority of the world's population by early adulthood (1). The virus establishes a lifelong infection with some cells being latently infected, a state in which the virus resides in a nonproductive form, while other cells show a low-level persistent productive infection in which the virus is not cleared from the organism and is periodically reactiv...

Interferon-responsive genes are targeted during the establishment of human cytomegalovirus latency

“…One function of IFI16 is the sensing of viral DNA and subsequent induction of Type I interferon or interleukin-1-beta 27,65 . While we do not currently rule out a potential role for IFI16-mediated sensing of HCMV during latency, we were more struck by the previous work identifying IFI16 as a modulator of host and viral transcription 32–34,36,37,66–71 . In particular, IFI16 is capable of activating the MIEP and driving IE gene expression within the first 6 hours of lytic infection of fibroblasts 32,34 , though at later times IFI16 blocks early and late gene expression 34,37 .…”

“…While we do not currently rule out a potential role for IFI16-mediated sensing of HCMV during latency, we were more struck by the previous work identifying IFI16 as a modulator of host and viral transcription 32–34,36,37,66–71 . In particular, IFI16 is capable of activating the MIEP and driving IE gene expression within the first 6 hours of lytic infection of fibroblasts 32,34 , though at later times IFI16 blocks early and late gene expression 34,37 . Given that a hallmark of HCMV latency is the suppression of IE gene expression 72 , we hypothesised that high levels of IFI16 might drive MIEP activity and IE gene expression in undifferentiated myeloid lineage cells.…”

“…Prior work has identified that IFI16 could activate IE gene expression in a UL83-dependent manner during lytic infection 32,37 , but since this tegument protein does not enter the nucleus in the CD34 + progenitor cell model of HCMV latency 74 , we hypothesised that IFI16 could activate the MIEP without additional virion components. To test this hypothesis, we utilized a THP-1 MIEP reporter system; THP-1 cells in which an integrated 1151 bp region of the MIEP drives the expression of eGFP 42 .…”

Tuning the Orchestra: HCMV vs. Innate Immunity

“…Thus, entrapping cytoplasmic IFI16 into virions might after all confer a fitness advantage to the virus (Cristea et al, 2010). However, more recent findings have shown that pp65 can also protect IFI16 from degradation, thereby favoring the inhibitory effect of this latter on the promoter region of UL54 (Biolatti et al, 2016). Interestingly, it has been recently demonstrated that IFI16 is rapidly targeted during the establishment of viral latency in a US28-dependent manner, but only in undifferentiated myeloid cells, a natural site of latent carriage (Elder et al, 2019).…”

Camouflage and interception: how pathogens evade detection by intracellular nucleic acid sensors

“…The deployment of different virulence factors to target several components of the DNA sensing pathway may be necessary to block the pathway more efficiently, and/or to inhibit some non‐overlapping functions of cGAS and IFI16 which have been observed during herpesvirus infection . Analogously, several different hCMV proteins inhibit DNA sensing: pUL31 binds to cGAS and dissociates it from DNA, pUL83 inhibits both cGAS and IFI16, pUL82 inhibits STING translocation, IE2 causes STING degradation, and US9 inactivates both MAVS and STING . Furthermore, pUL83 binds AIM2 and facilitates the degradation of AIM2‐driven inflammasomes .…”