Interferon-γ-inducible protein 16 (IFI16) is required for the maintenance of Epstein-Barr virus latency

Abstract Background Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It’s during the latent phase of EBV that all EBV-associated cancers, including Burkitt’s lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease arise. Interferon-γ-inducible protein 16 (IFI16) is a well-established innate immune sensor and viral transcriptional regulator involved in response to invading DNA viruses. During latency, IFI16 remains in the nucleus, in part bound to the EBV genome; however, neither its role in EBV lytic cycle or latency has been established. Methods Short interfering RNA against IFI16 and IFI16 overexpression were used to identify the role of IFI16 in the maintenance of EBV latency I. We also studied how induction of the lytic cycle affected IFI16 using the EBV positive, latently infected Akata or MUTU-1 cell lines. Akata cells were induced with TPA and MUTU-1 cells with TGF-β up to 96 h and changes in IFI16 protein were analyzed by Western blotting and immunofluorescence microscopy. To assess the mechanism of IFI16 decrease, EBV DNA replication and late lytic transcripts were blocked using the viral DNA polymerase inhibitor phosphonoacetic acid. Results Knockdown of IFI16 mRNA by siRNA resulted in enhanced levels of EBV lytic gene expression from all temporal gene classes, as well as an increase in the total EBV genome abundance, whereas overexpression of exogenous IFI16 reversed these effects. Furthermore, 96 h after induction of the lytic cycle with either TPA (Akata) or TGF-β (MUTU-1), IFI16 protein levels decreased up to 80% as compared to the EBV-negative cell line BJAB. Reduction in IFI16 was observed in cells expressing EBV lytic envelope glycoprotein. The decreased levels of IFI16 protein do not appear to be dependent on late lytic transcripts of EBV but suggest involvement of the immediate early, early, or a combination of both gene classes. Conclusions Reduction of IFI16 protein levels following lytic cycle induction, as well as reactivation from latency after IFI16 mRNA knockdown suggests that IFI16 is crucial for the maintenance of EBV latency. More importantly, these results identify IFI16 as a unique host factor protein involved in the EBV lifecycle, making it a potential therapeutic target to combat EBV-related malignancies

Kaposi's sarcoma-associated herpesvirus induces Nrf2 during de novo infection of endothelial cells to create a microenvironment conducive to infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies

ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and-III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

The IL-1β and type I interferon-β (IFN-β) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-β production during herpesvirus (KSHV, EBV, HSV-1) infections

LANA-1 puncta during infection of Nrf2-deficient cells.

<p>shRL- and shNrf2-transduced HMVEC-d cells were infected with KSHV (40 DNA copies/cell) for 24 hr prior to IFA analysis using a rabbit LANA-1 specific antibody (red). The vector containing shRL expresses the green fluorescent protein (GFP), which explains the green color of shRL cells that have been successfully transduced with the vector. <b>B</b>) Quantification of the number of KSHV+ (LANA-1+) cells in the shRL-transduced cells that expressed GFP (successfully transduced) and cells that did not (unsuccessfully transduced). Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 3, columns 1–2). <b>C</b>) Quantification of the number of KSHV+ (LANA-1+) cells in shRL vs. shNrf2 conditions. Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 4). <b>D</b>) Quantification of the number of LANA-1 dots/nucleus in shRL vs. shNrf2 conditions. Bars represent mean ± SD for three individual fields containing at least 10 cells each (panel A, row 4).</p

Effect of Nrf2 modulation on KSHV biology.

<p><b>A</b>) KSHV entry assay was performed on cells transduced with shRL or shNrf2 and infected with KSHV (20 DNA copies/cell). DNA real-time PCR was performed with ORF73 gene-specific primers, and the absolute KSHV copy number was calculated from a standard curve obtained by real-time PCR of standards with known concentration of ORF73. <b>B</b>) Starved HMVEC-d cells in a 48-well plate previously transduced with lentivirus vectors expressing either shGFP or shNrf2 were labeled with the ROS-measuring dye, CM-H₂DCFDA, and then infected with KSHV (40 DNA copies/cell) for the indicated time points prior to fluorescence measurement. Values indicate the mean ± SD for 3 independent replicates. <b>C</b>) KSHV nuclear delivery assay was performed on cells that were transduced with shRL or shNrf2 prior to infection with KSHV for 2 hr. Real-time PCR was performed using ORF73 gene-specific primers on DNA extracted from the nuclei of infected cells to determine the levels of viral DNA. The absolute copy number was calculated from a standard curve obtained by real-time PCR of standards with known concentrations of ORF73. Bars indicate mean ± SD for 3 independent replicates. <b>D and E</b>) Starved HMVEC-d cells transduced with either shRL or shNrf2 were infected for various times with KSHV (50 DNA copies/cell) and analyzed by one-step real-time PCR reaction and by WB using ORF50-specific primers and antibody, respectively. <b>F</b>) ORF73 (LANA-1) gene-specific primers were used to determine the expression levels of ORF73 from RNA as in panel 12D. The absolute copy number was calculated from a standard curve obtained by real-time PCR of RNA standards of ORF73 or ORF50 with known concentrations. Bars indicate mean copy number ± SD of 3 independent replicates. * = p<0.05, ns = p>0.05.</p

Nrf2 induction during UV-KSHV infection and during latent KSHV gene vFLIP overexpression.

<p><b>A</b>) Starved HMVEC-d cells were left uninfected, infected with functional KSHV, or infected with UV-treated KSHV for 2 hr (lanes 1–3) and 24 hr (lanes 4–6), and immunoblotted for pNrf2 and tNrf2. β-actin was used as loading control. Fold inductions normalized to β-actin and relative to the uninfected (U.I.) condition (arbitrarily set to 1) are indicated. <b>B–D</b>) HMVEC-d cells were transduced for 72 hr using vectors containing the four latent KSHV genes (ORF71/vFLIP, ORF72/vCyclin, ORF73/LANA-1 and ORFK12/Kaposin) and the level of relevant genes were determined by real-time RT-PCR. Bars indicate fold induction relative to pSIN A (arbitrarily set to 1) ± SD for 3 independent replicates. <b>E</b>) HEK293T cells transfected with control vector or with vector containing ORF71/vFLIP for 24 hr were assessed for levels of tNrf2 and pNrf2.</p

Colocalization of pNrf2 with KSHV genome and LANA-1.

<p><b>A</b>) Proximity ligation assay (PLA) on uninfected (U.I.) and KSHV-infected cells (20 DNA copies/cell) for 24 hr. Cells were incubated for 1 hr with antibodies against pNrf2 (rabbit) and LANA-1 (mouse monoclonal), washed, incubated for 1 hr with species-specific PLA probes and 2 additional oligonucleotides to facilitate the hybridization only in close proximity (<16 nm). A ligase was then added to join the two hybridized oligonucleotides to form a closed circle and initiate a rolling-circle amplification using the ligated circle as a template after adding an amplification solution to generate a concatemeric product extending from the oligonucleotide arm of the PLA probe. Lastly, a detection solution consisting of fluorescently-labeled oligonucleotides was added, and the labeled oligonucleotides were hybridized to the concatemeric products. The signal was detected as distinct fluorescent dots. Negative controls consisted of samples treated as described but with only secondary antibodies. Confocal microscopy was used for imaging. Red dots represent LANA-1 and pNrf2; blue staining = DAPI; white arrow = PLA dot [LANA-1+pNrf2] interaction. <b>B</b>) Quantification of the number of dots in the nuclei of infected HMVEC-d cells were obtained from 3 independent, representative fields, containing ∼30 cells each. <b>C</b>) HMVEC-d cells were infected with EdU-labeled KSHV and PLA for pNrf2 and LANA-1 (green dots) was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004460#ppat-1004460-g011" target="_blank">Figure 11A</a> prior to staining for EdU-KSHV (red). White arrows indicate the yellow colocalization spots between LANA-1+pNrf2 (PLA green spots) and EdU-KSHV genome (red). Blue arrows indicate the LANA-1+pNrf2 (PLA red spots) not colocalizing with EdU-KSHV genome.</p

Nuclear localization of Nrf2 during KSHV infection.

<p><b>A</b> and <b>B</b>) Nrf2 localization and levels during KSHV infection (20 DNA copies/cell) were visualized by IFA. Starved cells were infected with KSHV for 2 and 24 hr and stained for tNrf2 (<b>A</b>) or pNrf2 (<b>B</b>). DAPI was used to visualize the nuclei and merged tNrf2 or pNrf2/DAPI are shown in the third columns of the respective figure. Yellow square = enlarged area; red arrow = nuclear localization; white arrow = cytoplasmic localization. <b>C</b>) HMVEC-d cells were starved and infected as previously described, and the cytoplasmic and nuclear proteins were fractionated and then immunoblotted for pNrf2 and tNrf2. β-tubulin was used as a cytoplasmic purity control while TATA Binding protein (TBP) was used as a nuclear purity control. Fold inductions normalized to β-actin and relative to the uninfected (U.I.) condition (arbitrarily set to 1) are indicated.</p

Immunofluorescence analysis of Nrf2 levels in Kaposi's sarcoma skin lesions.

<p><b>A</b>) Healthy skin tissue (top row) and Kaposi's sarcoma skin tissue (bottom row) slides were assayed by immunofluorescence and incubated with rabbit anti-tNrf2 primary antibody and then with goat anti-rabbit (Alexa-Fluor 488-green) secondary antibody. DAPI was used to visualize the nuclei and the merged tNrf2/DAPI image is shown in the middle column. Yellow squares in the middle column indicate the area that has been enlarged in the right column. Red arrow = nucleus; white arrow = cytoplasm. <b>B</b>) Healthy skin (top two rows) and KS skin tissue (bottom row) were double-stained for LANA-1 (Alexa-Fluor 594- red) and host phosphorylated pNrf2 (Alexa-Fluor 488 – green). DAPI was used to visualize the nuclei, and the triple merge of LANA-1, pNrf2 and DAPI is shown in the third column. Yellow square = enlarged area. <b>C</b>) Quantitative representation of the colocalization of pNrf2, LANA-1 (KSHV+) and DAPI staining from the triple-merged figure of KS skin tissue in panel B. Cells that had detectable levels of staining of DAPI, pNrf2 and LANA-1 were considered as KSHV+/pNrf2+, while those staining only for DAPI were considered as KSHV−/pNrf2−. Bars indicate mean ± SD of 3 randomly selected fields containing at least 30 cells each (red boxes in Panel B). * = p<0.05. <b>D</b>) KS skin tissue containing an area dense in spindle cells (encircled by dashed line) and an area with low density of spindle cells (surrounding area). Yellow square indicates enlarged region; red arrow = triple colocalization. <b>E</b>) Venn diagram of the RGB color combination profiles.</p