Nuclear Innate Immune DNA Sensor IFI16 Is Degraded during Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus (KSHV): Role of IFI16 in Maintenance of KSHV Latency

Abstract

IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation.

Importance: Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.

FIG 1

RNA-Seq analysis of KSHV gene expression after IFI16 knockdown. (A, top) Immunoblot showing efficient knockdown of IFI16 in BCBL-1 cells 48 h and 96 h after transduction with a lentivirus carrying ShIFI16. (Middle) AIM2 immunoblot showing the absence of an off-target effect of the shRNA used. (B) Real-time RT-PCRs to determine IFI16 mRNA levels after 2 and 4 days of IFI16 knockdown. (C) Transcriptome analysis of KSHV after either induction with TPA or knockdown of IFI16 in BCBL-1 cells. Total RNAs were extracted 4 days after the indicated treatment and were subjected to cDNA library preparation using a QuantSeq 3′ mRNA-Seq Library Prep kit for Illumina sequencing. The libraries were sequenced by using HiSeq, and the sequences were mapped to the reference KSHV genome to determine the relative abundance of viral transcripts. Absolute values, calculated based upon the number of reads for each gene, were used for analysis of relative expression levels of KSHV genes as heat maps. (D) Next-generation RNA sequencing results from the three independent IFI16 knockdown experiments shown in panel C were compared to results for their respective control knockdowns (ShC), and the results are presented as relative fold changes in KSHV latent (a), immediate early (b), early (c), and late (d) gene expression ratios (ShIFI16/ShC). The dashed horizontal line indicates the 1.5-fold mark, set as the threshold. Values presented are the averages of results from three independent experiments.

FIG 2

Effect of IFI16 knockdown on KSHV lytic gene expression and viral DNA replication in latently KSHV-infected PEL cell lines. (A and C) Real-time RT-PCR using gene-specific primers for the four major classes of KSHV genes after ShIFI16-mediated IFI16 knockdown in BCBL-1 (A) and BC-3 (C) cells. mRNA levels were normalized against β-tubulin mRNA levels and are expressed as relative amounts compared to ShC (control) treatments (42). All cells were collected at 94 h postransduction. (B and D) Real-time RT-PCR showing IFI16 knockdown efficiencies. (E) Real-time PCR showing induction of KSHV DNA replication post-IFI16 knockdown in BCBL-1 cells. Treatment with TPA is shown as a positive control for lytic reactivation. Both TPA- and ShIFI16-treated cells were collected 96 h after treatment and transduction, respectively. Primers specific to the latent ORF73 gene were used, and the level of DNA was normalized against the β-tubulin gene level. (F) Same experiment as the one described above for panel E except that DNase treatment was performed prior to determination of genomic DNA copy numbers. Results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using the Student t test. **, P < 0.01; ***, P < 0.001.

FIG 3

Effect of IFI16 knockdown on TPA-induced KSHV gene expression and viral DNA replication in BCBL-1 cells. (A) BCBL-1 cells were lentivirally transduced with ShC or ShIFI16 as indicated. Forty-eight hours after knockdown, cells were treated with TPA (20 ng/ml), and total RNA was isolated at the indicated time points, as shown on the schematic diagram. Real-time RT-PCR using gene-specific primers for each of the four major classes of KSHV genes (La gene ORF73, IE gene ORF50, E gene K5, and L gene ORF25) was performed. mRNA levels were normalized against β-tubulin mRNA levels and are expressed as relative amounts compared to ShC (control) treatments. (B) Real-time DNA PCR showing relative KSHV genome copy numbers under the above-mentioned conditions. (C) Real-time RT-PCR showing IFI16 knockdown efficiencies at 0 and 96 h (see schematic in panel A) post-TPA treatment. Primers specific to the ORF73 gene were used, and the DNA level was normalized against the level of the β-tubulin gene. An immunoblot showing efficient knockdown of IFI16 at the indicated time points post-TPA treatment is also shown. All results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

FIG 4

Analysis of effects of IFI16 knockdown and TPA induction on KSHV lytic gene expression. (A) Immunofluorescence analysis of BCBL-1 cells after either IFI16 knockdown or TPA treatment (4 days). KSHV early lytic ORF59- and late gpK8.1A-specific monoclonal antibodies (49) were used to detect lytic reactivation (green fluorescence). BF, bright field. (B) Immunoblot of gpK8.1A after either IFI16 knockdown or TPA treatment (4 days). (C) Effect of IFI16 overexpression in BCBL-1 cells on TPA-induced KSHV lytic transcript expression. BCBL-1 cells were transduced with either a control lentivirus or an IFI16-overexpressing lentivirus. Twenty-four hours after lentiviral transduction, cells were induced with TPA for another 72 h. Total RNA was extracted, and mRNA levels of ORF50, ORF38, and IFI16 were analyzed by real-time RT-PCR using gene-specific primers. Results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using the Student t test. *, P < 0.05. (D) Immunoblot of IFI16 showing overexpression of IFI16 in BCBL-1 cells 24 h after transduction with an IFI16-overexpressing lentivirus or control lentivirus.

FIG 5

Effect of TPA induction on IFI16. (A) Immunoblotting of IFI16, RTA, and gpK8.1A in the KSHV-positive B-cell lines BCBL-1 and BC-3 after TPA or vehicle control induction for 48 and 96 h. UI, uninduced. (B) Immunoblotting of IFI16 in the KSHV-negative B-cell lines BJAB and Akata after induction with TPA or the vehicle control for 48 and 96 h. β-Actin was used to normalize band intensities of IFI16. (C) Real-time RT-PCR for IFI16 mRNA after TPA treatment for 2 and 4 days. Induction of the lytic ORF57 gene is shown to confirm lytic induction by TPA. Results are presented as means ± SD of data from three independent experiments.

FIG 6

Immunofluorescence analysis of IFI16 and KSHV lytic gpK8.1A expression in control and KSHV-positive cells after TPA induction. IFI16 and gpK8.1A in BJAB (A) and BCBL-1 (B) cells after TPA induction at the indicated time points are shown. UI, uninduced. Red and yellow arrows indicate IFI16 in the cytoplasm and nucleus, respectively. White arrows point to cells expressing gpK8.1A indicating lytic reactivation. These cells exhibit reduced IFI16 staining in the cytoplasm as well as in the nucleus, indicating IFI16 degradation. Enlarged images show IFI16 levels (green).

FIG 7

Effects of KSHV lytic reactivation induction by TPA-independent methods. (A) Immunofluorescence showing IFI16 and KSHV lytic gpK8.1A in TREX-BCBL-1 cells with or without doxycycline induction of RTA at the indicated times postinduction. White arrows indicate nuclear IFI16, and green arrows indicate cytoplasmic IFI16. Red arrows indicate gpK8.1A as a marker of KSHV lytic reactivation. (B) Immunoblot showing IFI16 levels after doxycycline induction in TREX-BCBL-1 cells. β-Actin was used to normalize band intensities of IFI16. (C) Immunoblotting of IFI16 and gpK8.1A in KSHV-positive BCBL-1 and KSHV-negative BJAB cells after treatment with neomycin or the vehicle control for 48 and 96 h. β-Actin was used to normalize band intensities of IFI16. UI, uninduced. (D) HMVEC-d cells were infected with KSHV (60 DNA copies/cell) or mock infected, incubated for 3 days to allow the establishment of latency, and treated with TPA or the vehicle control for the indicated time points, and IFI16 was probed by immunoblotting. IFI16 levels were normalized against the β-actin control.

FIG 8

Induction of the KSHV lytic cycle and ubiquitin-mediated IFI16 degradation in KSHV-positive PEL cell lines. (A) Immunoprecipitation (IP) was performed with an anti-IFI16 Ab under the indicated conditions, followed by immunoblotting with antiubiquitin antibody P4D1 (top) and an anti-IFI16 Ab (bottom). UI, uninduced; Ub, ubiquitin. (B) Proteasomal degradation was inhibited by using MG132. Shown are immunoblots of IFI16 and AIM2 in BCBL-1 and BJAB cells after treatment with TPA and MG132. (C) Treatment with 100 μg/ml PAA (4 days) was used to inhibit viral DNA synthesis and thus the expression of late lytic cycle proteins. Immunoblotting for IFI16 and gpK8.1A after the indicated treatments is shown. (D) Real-time DNA PCR using an ORF73 primer was done to assess the KSHV DNA replication-inhibitory function of PAA. (E) BCBL-1 cells were induced with TPA and 36 h later were treated with either 20 μM CHX (b and c) or the vehicle control DMSO (a) for the indicated time intervals. Immunoblots were performed for IFI16 and gpK8.1A. Time points of >8 h were toxic to cells. Results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 9

ChIP analysis of IFI16 in BCBL-1 and BC-3 cells. (A and B) IFI16 was immunoprecipitated from isolated nuclei of BCBL1 and BC-3 cells, and bound DNA was analyzed by real-time RT-PCR with primers specific to regions (∼100 to 200 bp) flanking the transcription start sites of the indicated genes. Relative promoter occupancy compared to IgG immunoprecipitation is presented. Results are presented as means ± SD of data from three independent experiments. (C) Agarose gel showing specific PCR amplification of the indicated KSHV promoter regions after IFI16 ChIP (BCBL-1 cells).

FIG 10

Luciferase reporter assay measuring IFI16 transcriptional repressor activity. (A) IFI16 was knocked down via lentivirus-mediated shRNA transduction in SLK cells. At 24 h post-KD, luciferase constructs of different KSHV gene promoters were transfected by using Lipofectamine. At 24 h posttransfection, the luciferase signal was quantitated by using a dual-luciferase assay and plotted. (B) Similar experiment as the one described above for panel A, using wild-type (wt) U2OS cells and CRISPR-IFI16 knockout U2OS clone 67 cells. (C) Similar experiment as the one described above for panel A, using SLK cells and luciferase constructs of different human gene promoters. (D) Luciferase assay after overexpression of IFI16 in U2OS clone 67 cells (IFI16 knockout). U2OS clone 67 cells were transfected with either an IFI16-overexpressing plasmid or a control plasmid, and after 24 h, luciferase constructs of different KSHV gene promoters were transfected. At 24 h posttransfection, the luciferase signal was quantitated by using a dual-luciferase assay and plotted. For all experiments, values are normalized to values for the renilla luciferase control. Results are presented as means ± SD of data from three independent experiments, and statistical analysis was done by using the Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.