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. Author manuscript; available in PMC: 2012 May 19.
Published in final edited form as: Cell Host Microbe. 2011 May 19;9(5):363–375. doi: 10.1016/j.chom.2011.04.008

Figure 6.

Figure 6

(A) Reconstitution of the IFI16 inflammasome. HEK293T cells were cotransfected with 0.5µg of pro-IL-1β, pro-caspase-1, ASC and IFI16 or AIM2 expressing plasmids as indicated. 24h post-transfection, the cells were infected with KSHV (30 DNA copies/cell) for 2h, washed and incubated for 24h. Protein lysates were examined for IL-1β maturation by immunoblotting. Tubulin was used as loading control. (B) Effect of KSHV genes on inflammasome induction. Empty vector, GFP or indicated KSHV genes were expressed in HMVEC-d cells via a lentivirus gene delivery system. Cells were also infected with KSHV for 2 h, washed and kept for 24h. Untransduced (UTr), transduced or KSHV infected cell lysates were examined for caspase-1 activation by immunoblotting with anti-caspase-1 antibodies. Actin was used as loading control. (C) FISH showing IFI16 and KSHV genome interaction. HMVEC-d cells were infected with KSHV (30 DNA copies/cell) for 2h, washed and incubated for the indicated time points. The cells were fixed, permeabilized and immuno-stained with mouse anti-IFI16 antibodies followed by donkey anti-mouse Alexa Fluor 594 secondary antibodies. The cells were then subjected to in situ hybridization with a spectrum green labeled whole KSHV genome probe. (D) HEK293T cells were cotransfected with 0.5mg pro-caspase-1, IFI16 and ASC-NLS expressing plasmids as indicated. 24h post-transfection, the cells were infected with KSHV (30 DNA copies/ cell) for 2h, washed, incubated for 24h and lysates were examined for caspase-1 activation by immunoblotting. (E) Schematic depicting a model for IFI16 mediated inflammasome activation in response to KSHV infection of endothelial cells. In uninfected endothelial cells, IFI16, ASC and caspase-1 are predominantly distributed in the nucleus. During de novo infection leading to the establishment of latency, IFI16, ASC and procaspase-1 form the multi-protein inflammasome complex in the nucleus leading to activation of caspase-1. The IFI16 containing inflammasome complex also migrates from the nucleus to the cytoplasm and forms peri-nuclear aggregates. Early during de novo infection, KSHV also induces the interaction between ASC and AIM2 possibly suggesting that the AIM2 inflammasome might be involved in sensing during the virus internalization process.