Kaposi's sarcoma-associated herpesvirus latency in endothelial and B cells activates gamma interferon-inducible protein 16-mediated inflammasomes

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) infections of endothelial and B cells are etiologically linked with Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma (PEL), respectively. KS endothelial and PEL B cells carry multiple copies of the nuclear episomal latent KSHV genome and secrete a variety of inflammatory cytokines, including interleukin-1β (IL-1β) and IL-18. The maturation of IL-1β and IL-18 depends upon active caspase-1, which is regulated by a multiprotein inflammasome complex induced by sensing of danger signals. During primary KSHV infection of endothelial cells, acting as a nuclear pattern recognition receptor, gamma interferon-inducible protein 16 (IFI16) colocalized with the KSHV genome in the nuclei and interacted with ASC and procaspase-1 to form a functional inflammasome (Kerur N et al., Cell Host Microbe 9:363-375, 2011). Here, we demonstrate that endothelial telomerase-immortalized human umbilical cells (TIVE) supporting KSHV stable latency (TIVE-LTC cells) and PEL (cavity-based B-cell lymphoma 1 [BCBL-1]) cells show evidence of inflammasome activation, such as the activation of caspase-1 and cleavage of pro-IL-1β and pro-IL-18. Interaction of ASC with IFI16 but not with AIM2 or NOD-like receptor P3 (NLRP3) was detected. The KSHV latency-associated viral FLIP (vFLIP) gene induced the expression of IL-1β, IL-18, and caspase-1 mRNAs in an NF-κB-dependent manner. IFI16 and cleaved IL-1β were detected in the exosomes released from BCBL-1 cells. Exosomal release could be a KSHV-mediated strategy to subvert IL-1β functions. In fluorescent in situ hybridization analyses, IFI16 colocalized with multiple copies of the KSHV genome in BCBL-1 cells. IFI16 colocalization with ASC was also detected in lung PEL sections from patients. Taken together, these findings demonstrated the constant sensing of the latent KSHV genome by IFI16-mediated innate defense and unraveled a potential mechanism of inflammation induction associated with KS and PEL lesions.

Fig 1

Activation of the inflammasome in cells latently infected with KSHV. (A) Lysates of endothelial cells latently infected with KSHV (TIVE-LTC), KSHV-negative endothelial (TIVE) cells, latent KSHV-positive B cells (BCBL-1), and KSHV-negative B cells (BJAB) were analyzed for procaspase-1 and activated caspase-1 (p20) proteins by immunoblot analysis. (B) BJAB and BCBL-1 cells were analyzed for IL-1β gene expression by real-time RT-PCR. Each bar represents the fold increase in gene expression ± SD of three independent experiments. Fold changes were calculated after normalization with expression of the 18S rRNA gene. (C and D) TIVE, TIVE-LTC, BJAB, and BCBL-1 cell lysates were analyzed for cleaved IL-1β (p17) protein (C) and for pro-IL-18 and cleaved IL-18 proteins (D) by immunoblot analysis. Tubulin and β-actin were used as loading controls.

Fig 2

KSHV latent protein vFLIP induces expression of genes associated with inflammasome activation. (A to C) Primary HMVEC-d cells were transduced with control (pSIN)-, ORF71-, ORF72-, ORF73-, ORF74-, or K12-expressing lentiviral vectors. At 72 h postransduction, expression of inflammasome-associated IL-1β, IL-18, and caspase-1 genes was analyzed by real-time RT-PCR. Each bar represents the fold increase in gene expression ± SD of three independent experiments. Fold changes were calculated by considering the levels of control transduced cells to be 1 after normalization with expression of the 18S rRNA gene. (D) Primary HMVEC-d cells transduced with control (pSIN), GFP-expressing, or vFLIP-expressing lentiviral vectors were examined after 72 h by phase-contrast and fluorescence microscopy. Magnification, ×40.

Fig 3

vFLIP-induced expression of inflammasome-associated genes is NF-κB dependent. (A) Primary HMVEC-d cells transduced with control (pSIN)-, GFP-, vFLIP-HA-wt-, and vFLIP-HA-A57L (mutant for NF-κB activation)-expressing lentiviral vectors were examined after 72 h by phase-contrast and fluorescence microscopy. Magnification, ×40. (B) Expression of HA-tagged vFLIP and its mutant protein in transduced HMVEC-d cells was confirmed by immunoblot analysis. Tubulin was used as a loading control. (C to G) Primary HMVEC-d cells were transduced with control (pSIN)-, GFP-, vFLIP-HA-wt-, or vFLIP-HA-A57L-expressing lentiviral vectors. At 72 h postransduction, expression of inflammasome-associated IL-1β-, IL-18-, caspase-1-, and NF-κB-induced TNF-α and IκBα genes was analyzed by real-time RT-PCR. Each bar represents the fold increase in gene expression ± SEM of three independent experiments. Fold changes were calculated by considering the levels of control transduced cells to be 1 after normalization with expression of the 18S rRNA gene.

Fig 4

ASC and caspase-1 interact and redistribute in the cytoplasm of cells latently infected with KSHV. TIVE and TIVE-LTC cells fixed with 2% paraformaldehyde (A and B) or BJAB and BCBL-1 cells fixed with ice-cold acetone (C and D) were subjected to immunofluorescence analysis. Cells were stained with anti-ASC and anti-caspase-1 antibodies and visualized by incubation with Alexa Fluor 488 (green) and Alexa Fluor 594 (red) secondary antibodies, respectively. Cell nuclei were stained with DAPI (blue). The boxed areas were enlarged and are shown in the rightmost panels. White and red arrows, colocalization of ASC and caspase-1 in the cytoplasm and nucleus, respectively. Image results are depicted from a representative field taken after three independent experiments were performed. Magnification, ×60.

Fig 5

IFI16 interacts with ASC and caspase-1 and translocates to the cytoplasm in cells latently infected with KSHV. (A) Whole-cell lysates of BJAB and BCBL-1 cells were examined for protein expression with IFI16, AIM2, NLRP3, ASC, and β-actin antibodies. (B) TIVE, TIVE-LTC, BJAB, and BCBL-1 cell lysates were immunoprecipitated with anti-ASC antibody and Western blotted for IFI16, AIM2, and NLRP3 proteins. (C) BJAB and BCBL-1 cell lysates were immunoprecipitated with anti-caspase-1 antibody and Western blotted for IFI16, AIM2, and NLRP3 proteins. ASC and caspase-1 IP blots are shown in Fig. 5. (B and C) (Bottom) IP efficiency. (D) BJAB and BJAB-KSHV cell lysates were immunoprecipitated with anti-caspase-1 antibody and Western blotted for IFI16, ASC, and caspase-1 proteins. Whole-cell lysates were also examined for protein expression with IFI16 and tubulin antibodies. (E and F) Expression and subcellular distribution of IFI16 were examined in nuclear and cytoplasmic fractions of TIVE and TIVE-LTC cells (E) and BJAB and BCBL-1 cells (F) by immunoblot analysis. TBP-1 and tubulin were used to demonstrate the purity and equal loading of nuclear and cytoplasmic fractions, respectively.

Fig 6

IFI16 colocalizes with ASC and redistributes in the cytoplasm of latently infected cells. (A and B) TIVE and TIVE-LTC cells fixed with 2% paraformaldehyde (A) or BJAB and BCBL-1 cells fixed with ice-cold acetone (B) were subjected to immunofluorescence analysis. Cells were reacted with anti-ASC and anti-IFI16 antibodies, washed, and incubated with Alexa Fluor 488 (green) and Alexa Fluor 594 (red) secondary antibodies, respectively. Cell nuclei were visualized by DAPI (blue). The boxed areas were enlarged, and the enlargements are shown in the far right panels. Red arrows, colocalization of ASC and IFI16. Image results are depicted from a representative field taken after three independent experiments were performed. Magnification, ×60.

Fig 7

Induction of the IFI16-ASC inflammasome and subcellular redistribution of IFI16-ASC during early and late stages of KSHV infection of endothelial cells. HMVEC-d cells were infected with KSHV (30 DNA copies per cell) for 2 h, uninternalized virus was removed by washing, and infected and uninfected cells were further incubated at 37°C for the indicated times. The cells were washed, fixed, permeabilized, and blocked with Image-iT FX signal enhancer. Cells were stained with anti-IFI16 and anti-ASC antibodies and visualized by incubation with Alexa Fluor 488 (green) and Alexa Fluor 594 (red) secondary antibodies, respectively. The image was merged with DAPI-stained nuclei. The boxed areas were enlarged, and the enlargements are presented in the rightmost panels. White arrows, colocalization. Image results are depicted from a representative field taken after three independent experiments were performed. UI, uninfected. Magnification, ×60.

Fig 8

Subcellular localization of ASC with Rab27a during latency establishment during primary KSHV infection and in latently infected PEL cells. Primary HMVEC-d cells either uninfected (A) or KSHV infected for 24 h (B) were fixed with 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 for 5 min, blocked with Image-iT FX signal enhancer, reacted with anti-ASC and anti-Rab27a antibodies, washed, and incubated with Alexa Fluor 488 (green) and Alexa Fluor 594 (red) secondary antibodies, respectively. (C and D) BJAB and BCBL-1 cells were fixed with ice-cold acetone, blocked with Image-iT FX signal enhancer, reacted with anti-ASC and anti-Rab27a antibodies, washed, and incubated with Alexa Fluor 994 (red) and Alexa Fluor 488 (green) secondary antibodies, respectively. Cell nuclei were visualized by DAPI (blue). The boxed areas were enlarged, and the enlargements are shown in the far right panels. White arrows, colocalization of ASC and Rab27a. Image results are depicted from a representative field taken after three independent experiments were performed. Magnification, ×60.

Fig 9

Detection of IFI16 and cleaved IL-1β in the exosomes released from cells latently infected with KSHV. (A) Exosomes released by BJAB and BCBL-1 cells in culture for 3 days were harvested and analyzed for the presence of IFI16, Rab27a, and cleaved IL-1β by Western blot analysis. The purity of exosome fractions was determined by the presence of multivesicular body-derived proteins Alix and Tsg101 and the absence of the ER protein calnexin. Ten micrograms of proteins was loaded per lane for Western blot analysis. Representative blots of 3 independent experiments are presented. (B) BJAB and BCBL-1 cells were immunostained for colocalization of the multivesicular body marker Tsg101 (green) and IFI16 (red). Cells were fixed, permeabilized, and blocked prior to immunostaining as described in the legend to Fig. 8. Alexa Fluor 594 (red) and Alexa Fluor 488 (green) secondary antibodies were used to detect IFI16 and Tsg101, respectively. Cell nuclei were visualized by DAPI (blue). White arrows, colocalization of IFI16 and Tsg101. Image results are depicted from a representative field taken after three independent experiments were performed. Magnification, ×60. (C) Whole-cell lysates of BJAB and BCBL-1 cells were examined for Tsg101 protein expression by immunoblot analysis. Tubulin was used as a loading control.

Fig 10

Colocalization of IFI16 with the KSHV genome in infected cell nuclei. (A) BJAB and BCBL-1 cells were fixed and immunostained with mouse anti-IFI16 antibody followed by anti-mouse Alexa Fluor 594 (red) secondary antibody. (B) Primary HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for 2 h, washed, and incubated for the indicated time points. The cells in panels A and B were then subjected to in situ hybridization with a Spectrum green-labeled whole-KSHV-genome probe and subjected to immunofluorescence image analysis. Cell nuclei were visualized by DAPI (blue). Yellow arrows, colocalization of the KSHV genome and IFI16. Magnification, ×60. (B) (Middle) Cells containing one copy of KSHV DNA shown by a single FISH spot with only one IFI16 colocalization spot; (bottom) a cell carrying multiple KSHV genomes with multiple IFI16 colocalization spots.

Fig 11

In vivo interaction of IFI16 and ASC in KSHV-infected solid PEL lesions. (A) Sections of lung from healthy individuals and solid PEL lesions from PEL patients were stained with mouse anti-IFI16 monoclonal and goat anti-ASC polyclonal antibodies and visualized by anti-goat Alexa Fluor 488 (green) and anti-mouse Alexa Fluor 594 (red) antibodies, respectively. Cell nuclei were visualized by DAPI (blue). The boxed areas were enlarged, and the enlargements are shown in the bottom panels (B). Yellow arrows point to the colocalization of ASC and IFI16. Image results are taken from a representative field (n = 3 healthy subject and patient tissue section samples). Magnification, ×60.

Fig 12

Schematic model depicting the sensing of the KSHV genome by IFI16 and inflammasome complex formation during latency. Results presented here demonstrate that KSHV episomal DNA is sensed in the infected cell during latency by innate DNA sensor IFI16 in the nucleus, leading to recruitment of adaptor protein ASC and pro-caspase-1 (Pro-Casp-1) to form an inflammasome complex. Inflammasome complex formation is followed by its translocation to the cytoplasm and activation of caspase-1, leading to cleavage of pro-IL-1β and pro-IL-18 to their mature forms. Both IFI16 and cleaved IL-1β are sorted and released into exosomal compartments, which could be a KSHV-mediated strategy to regulate their cognate and potential antiviral functions. A single IFI16-colocalizing spot in cells carrying a single copy of viral genome (Fig. 10B) and multiple IFI16-colocalizing spots in cells carrying multiple KSHV genomes (Fig. 10B), representing the sensing of individual genomes by IFI16, suggest that IFI16 must be sensing the KSHV genome continuously in the latently infected cells and thereby contributing to constitutive inflammasome activation. IFI16 interaction with the KSHV genome probably triggers changes that allow it to interact with ASC, leading to the formation of the inflammasome complex with ASC and caspase-1 and translocation to the cytoplasm, while a new IFI16 molecule probably comes into contact with the viral genome and thus results in a continuum of the above-described processes.