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. 2016 Aug 26;90(18):8238-50.
doi: 10.1128/JVI.00923-16. Print 2016 Sep 15.

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

Matteo Biolatti  1 , Valentina Dell'Oste  1 , Sara Pautasso  1 , Jens von Einem  2 , Manfred Marschall  3 , Bodo Plachter  4 , Marisa Gariglio  5 , Marco De Andrea  6 , Santo Landolfo  7
Affiliations

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

Matteo Biolatti et al. J Virol. .

Abstract

A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-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.

Importance: The 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.

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Figures

FIG 1
FIG 1
Effects of IFI16 overexpression on the activity of HCMV MIEP and UL54 gene promoter. (A) HFF cells were transiently electroporated with luciferase plasmids encoding MIEP or the UL54 gene promoter. Twenty-four hours later, the cells were transduced with AdVIFI16 (black bar) or AdVLacZ (gray bar) at an MOI of 50 or left uninfected (white bar). Afterwards, cells were infected with v65Rev (MOI of 1). Following a further 24 h, firefly and Renilla luciferase activities were measured. Luciferase activity in whole-cell lysates was normalized to Renilla luciferase activity. The mean value of v65Rev-infected cells (white bar) was arbitrarily defined as 1. Data represent the mean relative activities from three independent experiments, each performed in duplicate. A statistically significant difference compared to v65Rev-infected cells is indicated by ±SD (**, P < 0.01 by unpaired t test). RLU, relative light units. (B) HFFs were transduced with AdVIFI16 or AdVLacZ (MOI of 50) or were mock infected. After 24 h, cells were infected with v65Rev at an MOI of 1. Total cell extracts were prepared and subjected to Western blot analysis using anti-IFI16 polyclonal Ab. HCMV-IEA was employed as a positive control for viral infection, and α-tubulin served as the internal control.
FIG 2
FIG 2
pp65 and IFI16 are present at the MIEP and UL54 gene promoter. HFF cells were infected at an MOI of 1 with v65Rev or v65Stop virus and processed for ChIP assay 36 h later. Cells were cross-linked with formaldehyde, and then the IFI16, pp65, or appropriate control antibody (CTRL) immune complex was isolated by immunoprecipitation. (A) Immunoprecipitated proteins were examined by Western blotting with antibodies for IFI16 or pp65. (B to E) Purified ChIP DNA was analyzed by qPCR for the presence of the following specific DNA sequences: MIEP (B), UL54 (C), UL69 (D), and GAPDH (E). The mean value of control antibody (IgG) was arbitrarily defined as 1. A statistically significant difference compared to IgG is indicated by one (P < 0.05) or two (P < 0.01) asterisks (unpaired t test).
FIG 3
FIG 3
IFI16 requires the presence of pp65 for targeting MIEP and UL54 gene promoter. HFFs were electroporated with either pools of siRNAs targeting IFI16 (siIFI16) or scrambled control siRNA (siCTRL), and 24 h later they were infected with v65Rev or v65Stop at an MOI of 1 and examined at 36 hpi. (A) The efficiency of IFI16 depletion was assayed by Western blotting. (B and C) Cells were cross-linked with formaldehyde, and then IFI16 and pp65 immune complexes were isolated by immunoprecipitation. Purified ChIP DNA was analyzed by qPCR for the presence of the following specific DNA sequences: MIEP (B) and UL54 gene promoter (C). The mean value of control antibody (IgG) was arbitrarily defined as 1. A statistically significant difference compared to IgG is indicated by one (P < 0.05) or two (P < 0.01) asterisks (unpaired t test).
FIG 4
FIG 4
Interplay between IFI16 and pp65 regulates HCMV replication. (A) HFFs were electroporated with either pools of siRNAs targeting IFI16 (siIFI16) or scrambled control siRNA (siCTRL), and 24 h later they were infected with v65Rev or v65Stop at an MOI of 1 and examined at 48 hpi. The efficiency of IFI16 RNA depletion (A) and UL54 mRNA viral transcripts (B) was assayed by RT-qPCR. The relative levels of IFI16 and UL54 mRNA are normalized to the levels of cellular GAPDH. The data shown are averages from three experiments. Statistical significance is indicated by one (P < 0.05), two (P < 0.01), or three (P < 0.001) asterisks (unpaired t test). (C) Cells were infected as described for panel A. Viral DNA was isolated at 48 hpi and analyzed by qPCR. The primers amplified a segment of the IE1 gene to determine the number of viral DNA genomes per nanogram of cellular reference DNA (GAPDH gene). The data shown are averages (±SD) from three experiments, each performed in duplicate (*, P < 0.05; ***, P < 0.001; unpaired t test). (D) HFFs were electroporated with a pool of IFI16 small interfering RNAs (siIFI16) or scrambled control siRNA (siCTRL) and then infected with v65Rev or v65Stop (MOI of 0.1). The extent of virus replication was measured at the indicated times postinfection by titrating the infectivity of supernatants and cell suspension on HFFs by standard plaque assay. Results are expressed as means ± SD.
FIG 5
FIG 5
IFI16 protein is stably expressed during HCMV infection. (A) HFF cells were mock infected or infected with HSV-1 at an MOI of 1. (Left) Protein levels of IFI16 and ICP0 were assessed at different times postinfection (hpi) by Western blotting. (Right) IFI16 protein was subjected to densitometry and normalized to α-tubulin, serving as a loading control. The data show the mean values ± SD from three experiments (*, P < 0.05 by unpaired t test). (B) Kinetics analysis of IFI16 mRNA expression in HFFs upon HSV-1 infection at an MOI of 1 by RT-qPCR. IFI16 mRNA expression was normalized to that of GAPDH and is shown as the mean ± SD fold changes following HSV-1 versus mock infection (*, P < 0.05 by unpaired t test). (C) HFFs were mock infected or infected with HCMV v65Rev (MOI of 1). (Left) Lysates were prepared at the indicated time points and subjected to Western blot analysis for IFI16, IEA, and pp28. IFI16 protein was subjected to densitometry and normalized to α-tubulin, which served as a loading control. The data show the mean values ± SD from three experiments (***, P < 0.001 by unpaired t test). (D) Kinetics analysis of IFI16 mRNA expression in HFFs upon HCMV infection at an MOI of 1 by RT-qPCR. IFI16 mRNA expression was normalized to that of GAPDH and is shown as the mean ± SD fold change following HCMV versus mock infection (**, P < 0.05 by unpaired t test).
FIG 6
FIG 6
Confirmation of the pp65-IFI16 interaction. (A) Kinetics of IFI16-pp65 subcellular localization upon HCMV infection. HFF cells were mock infected or infected with HCMV v65Rev at an MOI of 1 for 48 or 144 h and then subjected to immunofluorescence analysis. pp65 (green) and IFI16 (red) were visualized using primary antibodies followed by secondary antibody staining in the presence of 10% HCMV-negative human serum. Nuclei were counterstained with DAPI (blue). (B to E) PLA was performed to detect protein-protein interactions using fluorescence microscopy. The signal was detected as distinct fluorescent dots in the Texas red channel when cells reacted with the indicated pairs of primary antibodies followed by PLA to assess the interactions between IFI16-IFI16 (B), IFI16-pp65 (C), IFI16-gB (D, upper), and IFI16-Sp3 (E, upper). (D and E, lower) HFF cells were infected with HCMV v65Rev virus at an MOI of 1 for 144 h and then subjected to immunofluorescence analysis. (F) Coimmunoprecipitation from virus-infected or mock-infected cell lysates. HFFs were infected with v65Rev virus (MOI of 1) and harvested at 48 or 144 hpi. Immunoprecipitations were performed using antibodies against IFI16 or pp65 and the appropriate control antibody (CTRL). Immunoprecipitated proteins were detected by Western blot analyses using antibodies against IFI16 and pp65. Nonimmunoprecipitated whole-cell extracts (Input) were immunoblotted using anti-IFI16 or anti-pp65 antibodies.
FIG 7
FIG 7
Stabilization of IFI16 in the presence of pp65. (A) HFFs were infected with wild-type, v65Rev, or v65Stop virus at an MOI of 1. (Upper) Lysates were prepared at the indicated time points and subjected to Western blot analysis for IFI16, IEA, pp65, and α-tubulin. (Lower) IFI16 was subjected to densitometry normalized to α-tubulin, which served as a loading control. Data present the means ± SD from three experiments (*, P < 0.05 by unpaired t test). (B) HFFs were infected with wild-type, v65Rev, and v65Stop viruses at an MOI of 1. Nuclear and cytoplasmic fractions were prepared at the indicated time points and subjected to Western blot analysis for IFI16 and pp65. α-Tubulin and TBP were used as purity and loading controls for the cytoplasmic and nuclear fractions, respectively. HCMV-IEA was employed as a positive control for viral infection. (C) Protein extracts were obtained from purified virions (v65Rev and v65Stop) and subjected to Western blot analysis using antibodies against IFI16 and MCP. MCP was employed to confirm infection and the isolation of virions. (D) HFFs were infected with either the v65Rev or v65Stop HCMV strain, and at 36 hpi they were incubated with cycloheximide (CHX) for 2 h. Lysates were subjected to Western blot analysis for IFI16, pp65, and α-tubulin, which served as a loading control.
FIG 8
FIG 8
IFI16 is retained in the nucleus during late stages of RV-VM1 infection. (A) Performance of the pp65 mutant RV-VM1 HCMV in fibroblasts compared with wild-type virus (RV-HB5). The arrows indicate large globular structures (LGS) in the nuclei typically associated with RV-VM1 replication. (B) HFF cells were mock infected or infected with RV-HB5 or RV-VM1 viruses at an MOI of 1 for 48 or 144 h and then subjected to immunofluorescence analysis. pp65 (green) and IFI16 (red) were visualized using primary antibodies followed by secondary antibody staining in the presence of 10% HCMV-negative human serum. Nuclei were counterstained with DAPI (blue). (C) PLA were performed to detect protein-protein interactions using fluorescence microscopy in HFF cells mock infected or infected with RV-HB5 or RV-VM1 virus at an MOI of 1 for 48 or 144 h. A positive signal was detected as distinct fluorescent dots in the Texas red channel when IFI16 and pp65 were in close proximity (∼40 nm). (D) HFFs were infected with RV-HB5 or RV-VM1 at an MOI of 1. Nuclear and cytoplasmic fractions were prepared at the indicated time points and subjected to Western blot analysis for IFI16, pp65, α-tubulin, and TBP. HCMV-IEA was employed as a positive control for viral infection.
FIG 9
FIG 9
Proposed model for the functional role of pp65 in modulating IFI16 activity during HCMV infection. IFI16 and pp65 cooperate at the level of viral genes, and the effect of this interaction (downregulation versus upregulation) depends on the viral promoter. In the early phases of infection, HCMV-pp65 hijacks IFI16 to activate MIEP expression; later on, IFI16 inhibits HCMV replication by blocking the UL54 gene promoter. Moreover, pp65 interacts with, stabilizes, and protects IFI16 from proteolytic degradation during HCMV infection. Finally, pp65 moves the protein into the cytoplasm, and then IFI16 becomes entrapped within newly assembled virions.
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