SUMMARY Inflammasomes are cytoplasmic sensors of foreign molecules, including pathogens, and function to induce caspase-1 activation and IL-1β cytokine maturation. Whether such a mechanism exists in the nucleus and is effective against nuclear replicating pathogens is unknown. Nuclear replicating herpesvirus KSHV is associated with Kaposi Sarcoma, an angioproliferative tumor characterized by an inflammatory microenvironment including IL-1β. We demonstrate that during KSHV infection of endothelial cells, interferon gamma-inducible protein 16 (IFI16) interacts with the adaptor molecule ASC and procaspase-1 to form a functional inflammasome. This complex was initially detected in the nucleus and subsequently in the peri-nuclear area. KSHV gene expression and/or latent KSHV genome is required for inflammasome activation and IFI16 colocalizes with the KSHV genome in the infected cell nucleus. Caspase-1 activation by KSHV was reduced by IFI16 and ASC silencing. Our studies reveal IFI16 as a nuclear pathogen sensor and demonstrate that the inflammasome also functions in the nucleus.

Kaposi's sarcoma (KS), an enigmatic endothelial cell vascular neoplasm, is characterized by the proliferation of spindle shaped endothelial cells, inflammatory cytokines (ICs), growth factors (GFs) and angiogenic factors. KSHV is etiologically linked to KS and expresses its latent genes in KS lesion endothelial cells. Primary infection of human micro vascular endothelial cells (HMVEC-d) results in the establishment of latent infection and reprogramming of host genes, and cyclooxygenase-2 (COX-2) is one of the highly up-regulated genes. Our previous study suggested a role for COX-2 in the establishment and maintenance of KSHV latency. Here, we examined the role of COX-2 in the induction of ICs, GFs, angiogenesis and invasive events occurring during KSHV de novo infection of endothelial cells. A significant amount of COX-2 was detected in KS tissue sections. Telomerase-immortalized human umbilical vein endothelial cells supporting KSHV stable latency (TIVE-LTC) expressed elevated levels of functional COX-2 and microsomal PGE2 synthase (m-PGES), and secreted the predominant eicosanoid inflammatory metabolite PGE2. Infected HMVEC-d and TIVE-LTC cells secreted a variety of ICs, GFs, angiogenic factors and matrix metalloproteinases (MMPs), which were significantly abrogated by COX-2 inhibition either by chemical inhibitors or by siRNA. The ability of these factors to induce tube formation of uninfected endothelial cells was also inhibited. PGE2, secreted early during KSHV infection, profoundly increased the adhesion of uninfected endothelial cells to fibronectin by activating the small G protein Rac1. COX-2 inhibition considerably reduced KSHV latent ORF73 gene expression and survival of TIVE-LTC cells. Collectively, these studies underscore the pivotal role of KSHV induced COX-2/PGE2 in creating KS lesion like microenvironment during de novo infection. Since COX-2 plays multiple roles in KSHV latent gene expression, which themselves are powerful mediators of cytokine induction, anti-apoptosis, cell survival and viral genome maintainence, effective inhibition of COX-2 via well-characterized clinically approved COX-2 inhibitors could potentially be used in treatment to control latent KSHV infection and ameliorate KS.

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In vitro Kaposi's sarcoma-associated herpesvirus (KSHV) infection of primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells is characterized

Infection of human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast (HFF) cells in vitro by Kaposi's sarcoma-associated herpesvirus (KSHV) provides an excellent in vitro model system to study viral latency. KSHV infection is characterized by the induction of preexisting host signal cascades; sustained expression of the latency-associated open reading frame 73 (ORF73) (LANA-1), ORF72, and K13 genes; transient expression of a limited number of lytic genes, including the lytic cycle switch ORF50 (replication and transcription activator) gene; and reprogramming of host transcriptional machinery regulating a variety of cellular processes, including several proinflammatory responses. The cyclooxygenase 2 (COX-2) gene was one of the host cell genes that was highly up-regulated at 2 and 4 h postinfection (p.i.) of HMVEC-d and HFF cells (P. P. Naranatt, H. H. Krishnan, S. R. Svojanovsky, C. Bloomer, S. Mathur, and B. Chandran, Cancer Res. 64:72-84, 2004). Since COX-2 is an important mediator of inflammatory and angiogenic responses, here, using real-time PCR, Western blot, and immunofluorescence assays, we characterized the COX-2 stimulation and its role in KSHV infection. KSHV induced a robust COX-2 expression, which reached a maximum at 2 h p.i. in HMVEC-d cells and at 8 h p.i. in HFF cells, and significantly higher levels were continuously detected for up to 72 h p.i. Constitutive COX-1 protein levels were not modulated by KSHV infection. Moderate levels of COX-2 were also induced by UV-irradiated KSHV and by envelope glycoproteins gB and gpK8.1A; however, viral gene expression appears to be essential for the increased COX-2 induction. High levels of prostaglandin E2 (PGE2), a COX-2 product, were released in the culture supernatant medium of infected cells. PGE2 synthase, catalyzing the biosynthesis of PGE2, also increased upon infection and inhibition of COX-2 by NS-398, and indomethacin drastically reduced the levels of PGE2 and PGE2 synthase. COX-2 inhibition did not affect KSHV binding, internalization of virus, or the trafficking to the infected cell nuclei. However, latent ORF73 gene expression and ORF73 promoter activity were significantly reduced by COX-2 inhibitors, and this inhibition was relieved by exogenous supplementation with PGE2. In contrast, lytic ORF50 gene expression and ORF50 promoter activity were unaffected. These studies demonstrate that COX-2 and PGE2 play roles in facilitating latent viral gene expression and the establishment and maintenance of latency and suggest that KSHV has evolved to utilize the inflammatory responses induced during infection of endothelial cells for the maintenance of viral latent gene expression.