Figure 1.
Immunofluorescence analysis of Nrf2 levels in Kaposi's sarcoma skin lesions.
A) 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) 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. C) 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. D) 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. E) Venn diagram of the RGB color combination profiles.
Figure 2.
Induction of Nrf2 activity during de novo KSHV infection.
A) Primary endothelial (HMVEC-d) cells starved for a total of 8 hr were infected with KSHV (20 DNA copies/cell) for the indicated time points and immunoblotted with pNrf2 and tNrf2 antibodies. 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) Starved HMVEC-d cells were treated with 100 µM H2O2 for 15 min prior to western blot analysis for pNrf2 and tNrf2. C) Graphical representation of the ratio of pNrf2/tNrf2 fold induction as observed in the Western blot illustrated in figure 1A. Dashed line = hypothetical line depicting constitutive phosphorylation; red line = actual ratio of pNrf2/tNrf2 during KSHV infection; orange asterisk = ratio of pNrf2/tNrf2 from H2O2 treatment. D) Starved HMVEC-d cells were infected with KSHV for 2 hr, incubated for 16 hr in growth factor-supplied media prior to additional starvation for 8 hr, at which point they were immunoblotted for pNrf2 and tNrf2.
Figure 3.
Nuclear localization of Nrf2 during KSHV infection.
A and 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 (A) or pNrf2 (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. C) 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.
Figure 4.
Effect of KSHV ROS induction on Nrf2-Keap1 interaction and Nrf2 ubiquitination.
A) Real-time RT-PCR analysis of Nrf2 mRNA measured at various times post-infection of HMVEC-d cells. β-tubulin was used as an endogenous house-keeping gene. Each point represents fold induction compared to uninfected cells (Unt; arbitrarily set to 1) ± SD for 4 independent experiments. * = p<0.05. B) Starved HMVEC-d cells were treated with NAC (10 mM) or PDTC (100 µM) for 2 hr prior to infection with KSHV (20 DNA copies/cell) for an additional 2 hr before immunoblot analysis for tNrf2 and pNrf2. NAC = N-Acetylcysteine; PDTC = pyrrolidine dithiocarbamate. C) Starved HMVEC-d cells were treated with increasing concentration of DPI (0–50 µM) for 2 hr prior to infection with KSHV (20 DNA copies/cell) for an additional 2 hr before immunoblot analysis. DPI – Diphenyleneiodonium. D) Infected HMVEC-d cells were immunoblotted for pNrf2, tNrf2 and Keap1. Fold inductions normalized to β-actin and relative to the uninfected (U.I.) condition (arbitrarily set to 1) are indicated. E) Real-time RT-PCR analysis of Keap1 mRNA measured 24 hr p.i. from HMVEC-d cells infected with KSHV (20 DNA copies/cell). β-tubulin was used as endogenous house-keeping control. Bars represent fold change ± SD for 3 independent experiments. * = p<0.05. F) HMVEC-d cells were infected for the indicated time points and lysed with NETM buffer to isolate whole cell protein. Equal amounts of protein (200 µg/condition) were immunoprecipitated using anti-Keap1 or anti-Nrf2 antibody O/N at 4°C and immunoblotted for tNrf2 and Keap1 or Lysine-48-linked Ubiquitin (Ub-K48) and tNrf2, respectively. Bottom three panels indicate the whole cell lysate levels of Nrf2, Keap1 and β-tubulin. G) The pull-down results were normalized to either whole cell lysate (input) β-tubulin (black bars) or Nrf2 (white bars), and fold inductions relative to the uninfected conditions (U.I. - arbitrarily set to 1) are indicated. The Keap1-Nrf2 interaction is shown in the top graph, and the Nrf2 ubiquitination levels are shown in the bottom graph.
Figure 5.
Inhibition of virus binding, Src, PI3-K and PKC-ζ and their effect on KSHV-mediated Nrf2 induction.
A) Starved HMVEC-d cells were infected with either functional KSHV (lane 2) or with heparin-treated (20 µg/ml for 1 hr) KSHV (lane 3). Media alone (lane 1) or 20 µg/ml of heparin alone (lane 4) were used to determine if heparin had any effect on Nrf2 activity. B–E) HMVEC-d cells were starved for 8 hr, during which time they were treated with Src inhibitor PP2 (B), PI3-K inhibitors LY294002 (irreversible) and Wortmannin (reversible) (C), NFκB inihibitor Bay-11-7082 (D), PLC-γ inhibitor U-73122 (E), and Myristoylated PKC-ζ pseudosubstrate to inhibit PKC-ζ (consists of amino acids 113–129 of the substrate region) (F). One hour after drug or mock (DMSO) treatment, cells were infected for 30 min with KSHV (20 DNA copies/cell). Protein lysates were immunoblotted for pNrf2 and tNrf2, while β-tubulin/β-actin was used as a loading control. Fold inductions normalized to β-tubulin/β-actin and relative to the uninfected (U.I.) condition (arbitrarily set to 1) are indicated. G) HMVEC-d cells were treated with inhibitors as in panels B–F, infected with KSHV (20 DNA copies/cell) for 4 hr to allow Nrf2-dependent gene transcription to take place, and subjected to real-time RT-PCR for the Nrf2 target gene NQO1 [NAD(P)H quinone oxidase 1]. Bars indicate the fold change compared to the uninfected/untreated condition (arbitrarily set to 1) ± SD for 3 independent replicates. * = p<0.05.
Figure 6.
Lentiviral knockdown of Nrf2 and the effects on target genes during KSHV infection.
A) Top: Nrf2 ELISA was used to quantify the DNA (ARE)-binding affinity of Nrf2 during KSHV infection (20 DNA copies/cell). Nuclear proteins from infected and uninfected cells were quantified using a BCA assay, and equal amounts of protein (15 µg) were assayed using the TransAM Nrf2 ELISA Kit. The OD450, measuring the binding of Nrf2, in the uninfected condition (U.I.) was arbitrarily set to 1, and the bars indicate mean fold induction ± SD for two independent replicates. 30 µg of MCF7 nuclear extract was used as a positive control for Nrf2 activity. Bottom: Western blot for pNrf2 in the nuclear fraction was performed for quality control. β-tubulin was used as a cytoplasmic control, TATA Binding protein (TBP) was used as a nuclear control, and β-actin was used as loading control. B) Real-time RT-PCR analysis assessing Nrf2 mRNA levels in HMVEC-d cells transduced with lentiviral vectors expressing short-hairpin RNA against Renilla luciferase (shRL – control) or against Nrf2 (shNrf2) for at least 24 hr. The uninfected (U.I.) shRL condition was arbitrarily set to 1 and the bars indicate mean fold induction ± SD for 4 independent experiments. C) Representative Western blot analysis assessing Nrf2 knockdown in HMVEC-d cells transduced with lentiviral vectors expressing shRL or shNrf2 for at least 24 hr, and infected with KSHV (20 DNA copies/cell) for the indicated time points. NQO1 induction was used to determine the activity of Nrf2 transcriptional activity during KSHV infection. β-actin was used as a loading control. D) Real-time RT-PCR analysis of Nrf2 target genes involved in ROS homeostasis. β-tubulin was used as an endogenous control, the U.I. shRL cells were arbitrarily set to 1, and the bars indicate mean fold induction ± SD for 4 independent experiments. * = p<0.05. (NQO1 = NAD(P)H quinone oxidase 1; GCS = Gamma-glutamylcysteine synthase; HO1 = Heme oxygenase 1).
Figure 7.
VEGF activity during KSHV infection and its dependence on Nrf2.
A) Starved HMVEC-d cells were infected with KSHV (20 DNA copies/cell) for 8 hr (left) and 24 hr (right) prior to real-time RT-PCR analysis for 3 members of the vascular endothelial growth factor (VEGF) family: VEGF-A (red), C (white) and D (black). β-tubulin was used as an endogenous house-keeping control and gene expression of uninfected cells (not shown) was arbitrarily set to 1 as depicted by the horizontal dashed line, while the bars indicate mean fold induction ± SD for 3 independent experiments. * = p<0.05 for each time/gene when compared to the respective U.I. control. B) VEGF165 ELISA (A-isoform) (Quantikine Kit – R&D) was performed on starved HMVEC-d cells infected with KSHV (20 DNA copies/cell) for 8 hr prior to supernatant collection and quantification of absolute levels using a standard curve. C) VEGF165 ELISA was performed in shRL and shNrf2 cells infected with KSHV (20 DNA copies/cell) for the indicated times post-infection. For panels B and C, values indicate the mean of supernatant protein concentrations ± SD for 3 independent replicates. D) Real-time RT-PCR of Nrf2 mRNA in conditions from panel C, to determine the efficiency of lentiviral knockdown of Nrf2. * = p<0.05.
Figure 8.
KSHV infection and the Nrf2-COX-2-PGE2 loop.
A) HMVEC-d cells were transduced with lentiviral vectors expressing shRL or shNrf2 for 24 hr, and then infected with KSHV (20 DNA copies/cell) for the indicated time points prior to real-time RT-PCR analysis for COX-2 (Cyclooxygenase-2). The U.I. shRL condition was arbitrarily set to 1 and the bars indicate fold induction ± SD for 5 independent experiments. β-tubulin was used as an endogenous control. * = p<0.05. B) shRL- and shNrf2-transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell) for the indicated time points and immunoblotted for COX-2 and COX-1. β-actin was used as a loading control and the fold induction relative to each U.I. condition (arbitrarily set to 1) is indicated. C) The human COX-2 promoter sequence (2500 base-pairs upstream of the transcriptional start site) was obtained from ensemble.org (Accession #: ENSG00000095303). Antioxidant Response Element (ARE) consensus sequences are indicated with red arrows, whereas ARE-like sequences are identified with grey arrows. D) Starved HMVEC-d cells were treated with an increasing concentration (0.1–100 µM) of Prostaglandin E2 (PGE2) for 4 hr prior to immunoblot analysis for pNrf2 and tNrf2. β-tubulin was used as loading control. E) Starved HMVEC-d cells were treated with 10 µM PGE2 for 4 hr prior to RNA extraction and real-time RT-PCR analysis of Nrf2 gene expression levels. β-tubulin was used as an endogenous control. The uninfected condition (U.I.) was arbitrarily set to 1, and the points indicate fold induction ± SD for 3 different replicates. ns = p>0.05. F) Starved HMVEC-d cells were pretreated with 10 µM Myristoylated-PKC-ζ pseudosubstrate for 1 hr before addition of PGE2 for an additional 4 hr and analysis by immunoblot assay for pNrf2 and tNrf2. β-tubulin was used as a loading control and the fold induction relative to each U.I. condition (arbitrarily set to 1) is indicated. G) HMVEC-d cells were pretreated with mock inhibitor or Celecoxib (10 µg/ml) for 2 hr prior to KSHV infection (20 DNA copies/cell) for the indicated time points and immunoblotted for pNrf2 and tNrf2. H) Graphical representation of the fold induction of pNrf2 and tNrf2 levels in Celecoxib- and mock (DMSO)-treated cells. Fold induction was normalized to β-actin and is relative to their respective U.I. condition, which was arbitrarily set to 1.
Figure 9.
Nrf2 induction during UV-KSHV infection and during latent KSHV gene vFLIP overexpression.
A) 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–D) 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. E) HEK293T cells transfected with control vector or with vector containing ORF71/vFLIP for 24 hr were assessed for levels of tNrf2 and pNrf2.
Figure 10.
Effect of KSHV binding and gene expression on virus-mediated Nrf2 induction.
A) Deconvoluted IFA imaging of HMVEC-d cells infected with BrdU-labeled genome containing KSHV for 24 hr, and stained with rabbit anti-pNrf2 primary antibody and goat anti-rabbit (Alexa-Fluor 488-green) secondary antibody, and with mouse anti-BrdU primary antibody and goat anti-mouse (Alexa-Fluor 594-red) secondary antibody. Yellow square = enlarged area; red arrows = infected cell; white arrows = uninfected cells with low pNrf2; blue arrows = uninfected cell with high nuclear pNrf2; blue staining = DAPI. Yellow arrows = colocalization of pNrf2 and BrdU-labeled KSHV genome. B) Confocal imaging of two HMVEC-d cells for pNrf2, BrdU-labeled KSHV genome and DAPI. Dashed white line separates the field in two, the top field containing an uninfected cell while the bottom field containing a 24 hr KSHV-infected cell. C) Confocal imaging on three separate stacks of the same cell infected with BrdU-labeled KSHV for 24 hr. Triple-merged figures are shown on the bottom-most row, and representative colocalization figures are enlarged in the yellow boxes.
Figure 11.
Colocalization of pNrf2 with KSHV genome and LANA-1.
A) 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) 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. C) HMVEC-d cells were infected with EdU-labeled KSHV and PLA for pNrf2 and LANA-1 (green dots) was performed as described in Figure 11A 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.
Figure 12.
Effect of Nrf2 modulation on KSHV biology.
A) 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) 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. C) 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. D and E) 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. F) 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.
Figure 13.
LANA-1 puncta during infection of Nrf2-deficient cells.
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) 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). C) 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). D) 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).
Figure 14.
Schematic diagram showing Nrf2 induction and consequences during de novo KSHV infection of endothelial cells.
Phase I. KSHV binding to cellular receptors induces signaling that involves multiple kinases and ROS. ROS induction inhibits degradation of Nrf2 by the Keap1-Cul3 ubiquitination axis, allowing for its rapid accumulation inside the cells. Signaling kinases induced by the interaction of KSHV with its cell-surface receptors to facilitate virus entry and gene expression also mediate Nrf2 phosphorylation, which serves to further stabilize Nrf2 by decreasing its association with Keap1 as well as by increasing its nuclear localization. Nrf2 nuclear localization induces expression of well-known anti-oxidative target genes as well as additional genes such as COX-2, Bcl-2 and VEGF. Phase II. As the original signaling wanes, the Nrf2-mediated COX-2 induction releases PGE2 in the surrounding environment, which in turn induces Nrf2 activation via autocrine and paracrine signaling pathways involving PKC-ζ. This establishes a feed-forward loop where Nrf2 activation induces COX-2 expression, which releases PGE2 and mediates further Nrf2 activation. As latency initiates, vFLIP expression further fuels the COX-2/PGE2 axis, and likely other pathways, to augment Nrf2 activity. Phase III. As virus establishes latency, Nrf2 activity appears to be important at several levels in its biology, as its inhibition upregulates ORF73 expression and decreases ORF50 and other lytic expression. Nrf2 also colocalizes with the major latency protein, LANA-1, as determined by PLA, and both colocalized with the KSHV genome, probably modulating the expression of viral and host genes.