Our studies suggest that KSHV induces the inflammasome via a previously unknown pathway and IFI16 possesses, hitherto not identified, properties of interaction with ASC to form a caspase-1 activating molecular platform. IFI16, a resident nuclear protein, is known for its role in cell cycle regulation and DNA damage responses (Aglipay et al., 2003
; Fujiuchi et al., 2004
Studies by Bryan et al., (Bryan et al., 2009
) demonstrated that inflammasome activation in THP1 cells by bacterial RNA results in the redistribution of ASC from the nucleus to the cytoplasm and colocalization with caspase-1 and NLRP3 in the peri-nuclear area. Our demonstration of active caspase-1 in the nuclear fractions of infected HMVEC-d cells at 2h and 8h p.i. () as well as activation of caspase-1 in the ASC-NLS containing IFI16 inflammasome in HEK293T cells () suggest that inflammasome assembly can also occur in the nucleus. However, at later times of KSHV infection, the majority of activated caspase-1 was observed in the cytoplasmic fractions. This shift in the caspase-1 activation from nucleus to cytoplasm coupled with nuclear to cytoplasmic redistribution of ASC, caspase-1 and IFI16 indicate a higher order of complexity involved in the regulation of caspase-1 activation (). Such mechanism may help preventing excessive caspase-1 activation and/or to avert unwanted caspase-1 activity in the nucleus.
Cytosolic sensors like AIM2 and NLRP3 have been shown to activate inflammasomes in response to infection by bacteria, DNA and RNA viruses (Burckstummer et al., 2009
; Fernandes-Alnemri et al., 2009
; Hornung et al., 2009
; Kim et al., 2010
; Muruve et al., 2008
; Rathinam et al., 2010
; Schroder et al., 2009
). During in vitro infection of endothelial cells, KSHV does not undergo a productive cycle, instead it enters into a latency program and the genome persists in the nuclei as an episome tethered to host chromatin. Interestingly, UV-inactivated KSHV did not to induce significant caspase-1 activation at 48h p.i. () prompting us to surmise that establishment of latency is critical for continued inflammasome induction. However, none of the KSHV latency associated genes were able to induce caspase-1 activation suggesting that the KSHV latency genes are not directly involved in inflammasome activation. Additionally, colocalization of IFI16 with the KSHV genome in the infected cell nuclei () suggests that IFI16 functions as a sensor of pathogen associated molecular pattern (PAMP) within the nucleus. Our studies showed that silencing IFI16 inhibited inflammasome activation without affecting AIM2 or NLRP3 pathways. Furthermore, IFA staining and co-IP studies showed ASC, IFI16 and cascase-1 interaction. Additionally, IFI16 did not mediate caspase-1 activation during vaccinia virus infection that replicates in the cytoplasm. Taken together, these observations demonstrate that IFI16 along with ASC and caspase-1 forms a functional inflammasome in response to KSHV infection in the nucleus.
Earlier studies did not detect interaction between ASC and IFI16 (Burckstummer et al., 2009
; Fernandes-Alnemri et al., 2009
; Hornung et al., 2009
). These studies were aimed at identifying a protein that can detect cytoplasmic DNA and used transfected cytoplasmic DNA as a trigger to examine ASC interaction with IFI16 and other HIN-200 members. Therefore, nuclear IFI16 or other nuclear HIN-200 proteins may not have any role in this context. ASC-IFI16 interaction becomes detectable in the presence of latent KSHV infection with a little constitutive interaction in uninfected cells which suggests the requirement for an appropriate stimulus in the nucleus. Hence, the lack of an appropriate trigger, such as nuclear viral DNA in the above studies, could be one of the reasons for not detecting IFI16 in inflammasome activation. This is very well supported by our inflammasome reconstitution studies in HEK293T cells. When IFI16 was expressed along with other inflammasome components, elevated IL-1β maturation was observed only upon infection with KSHV (, lane 3) whereas over expression without infection induced substantially lower levels of IL-1β maturation (, lane 2). Differential inflammasome activation in response to adenoviral DNA also supports the above cited possibilities (Muruve et al., 2008
). The inflammasome is activated via NLRP3 when adenovirus DNA is delivered by infection or transduction, while NLRP3 is not required in response to adenovirus DNA delivered via lipid-mediated transfection suggesting that virus infection is sensed in a different way than transfected DNA.
Sub-cellular distribution of ectopically expressed caspase-1 depends on the cell type. Ectopically expressed caspase-1 was predominantly cytoplasmic with little nuclear distribution in HEK293 and Jurkat cells, whereas in HeLa cells the expression was predominantly nuclear (Shikama et al., 2001
). Similarly, ASC isoforms vary in their sub-cellular distribution as well as in their ability to form functional inflammasomes (Bryan et al., 2010
). Hence, it is possible that differences might exist between endothelial cells, macrophages and THP1 cells with respect to inflammasome activation.
The large amount of viral genome in the infected cell nuclei is often sensed as abnormal extra-chromosomal DNA leading to activation of DNA damage response (DDR) signaling pathways (Schwartz et al., 2009
; Weitzman et al., 2004
). IFI16 has been shown to be a part of the large multi-protein BRCA1-associated genome surveillance complex (BASC) that is involved in the DDR (Aglipay et al., 2003
; Wang et al., 2000
). Notably, IFI16 undergoes nuclear to cytoplasmic redistribution following exposure to ultraviolet-B (UVB) radiation (Costa et al., 2011
). Similar sub-cellular redistribution of IFI16 following KSHV infection suggests that the latent KSHV genome in the infected cell nucleus is recognized as DNA damage similar to UVB exposure. Furthermore, no such redistribution of IFI16 was observed upon infection by cytoplasimc vaccinia virus. Thus, IFI16 might function as a double-edged sword sensing the KSHV genome in the nucleus as ‘damage’ and inducing the inflammasome. IFI16 with its dual potential to bind DNA (via HIN-200) and ASC (via PYD) may be strategically localized in the cell nucleus to detect abnormal/non-self genetic material and respond by inducing the inflammasome.
In uninfected cells, IFI16, ASC and caspase-1 proteins are predominantly localized to the nucleus. During early time of KSHV infection (2h), these proteins formed a complex in the nucleus, and at later time points these proteins colocalized in the perinuclear area. It is possible that IFI16 directly or indirectly through the involvement of some other proteins is sensing the KSHV genome in the nucleus. This sensing might result in the activation of signaling pathways to induce the nuclear export of ASC/Caspase-1/IFI16 as complex or individual proteins, which assemble into functional inflammasome complex in the perinuclear area (). Further studies are required to understand the reason for sub-cellular redistribution upon sensing the stimulus.
The de novo infected endothelial cells as well as in KS lesions show signs of persistent DNA damage response as demonstrated by the presence of activated ATM– Chk2 signaling pathways, γ-H2AX and p53 binding protein-1 (p53BP1) (Koopal et al., 2007
). If the extent of DNA damage is irreparable as seen in the latent KSHV infection (Koopal et al., 2007
), the cells may maintain a chronic, low level DDR. This persistent DDR is known to induce the robust secretion of inflammatory cytokines called Senescence-Associated Secretory Phenotype (SASP) (Coppe et al., 2010
; Rodier et al., 2009
). Knockdown of upstream components of the DDR such as ATM, NBS1, or CHK2, has been shown to prevent the induction of inflammatory cytokines (Rodier et al., 2009
). Additional studies are required to study whether IFI16 directly recognizes KSHV genome or acts as downstream link in the DDR response initiated by Mre/Rad50/NBS1 and ATM.
Although the IFI16 inflammasome can be viewed as a host intrinsic response to KSHV infection, it is possible that KSHV has evolved to usurp this mechanism for establishing latency since IL-1β has been shown to up-regulate KSHV latent gene expression (Yu et al., 1999
). However, the levels of IL-1β secretion from KSHV infected endothelial cells were low compared IL-1β secretion from THP1 cells. Hence, further studies are required to establish the biological significance of IL-1β secretion from KSHV infected endothelial cells as well as whether IFI16 is involved in inflammasome induction during primary as well as latent infection of other cell types including THP1 cells and lymphoma cells. A recent study has demonstrated that KSHV tegument protein ORF63 is an NLR homolog which can inhibit inflammasome activation by binding to NLRP1 and NLRP3 (Gregory et al., 2011
). This study also demonstrated that inflammasome activation suppresses KSHV reactivation from latency suggesting that inflammasome activation and IL-1β mediated signaling facilitates KSHV latency. During primary KSHV infection and internalization, KSHV tegument ORF63 protein might be binding to NLRP3 and NLRP1 to prevent the detrimental effects of inflammasome activation. This could also be a potential reason for the absence of NLRP3 in our IP reactions (). Thus KSHV might have evolved to hijack ORF63 mediated phenomenon to establish latency during primary infection. However, on the other hand, IFI16 mediated inflammasome activation after delivering the viral genome to the nucleus could be aiding the virus to establish latency. Further studies are needed to understand the role of IFI16 in KSHV induced inflammasome in other cell types including monocytes and KSHV and EBV transformed B cell lymphoma cell lines.
Studies demonstrated that IFI16 is critical for the interferon-β response upon exposure to intracellular cytoplasmic DNA and HSV-1 infection (Unterholzner et al., 2010
). IFI16 was directly associated with IFN-β-inducing viral DNA motifs and STING involved in IFN-β responses to DNA. These authors thus identified IFI16 as a PYD-HIN protein involved in IFN-β induction and labeled IFI16 and AIM2 as a new family of innate DNA sensors “'AIM2-like receptors' (ALRs)”. The involvement of IFI16 in inflammasome induction was not examined in this study. Our findings demonstrate that IFI16 induces inflammasome activation in response to KSHV infection and identifies IFI16 as a nuclear pathogen sensing molecule (receptor) analogous to cytoplasmic PRR inflammasomes. It is possible that NLRP1, NLRP3, NLRC4 and AIM2 might have evolved to detect cytoplasmic pathogen cues, while IFI16 might have evolved to detect nuclear pathogens. Additional studies are required to decipher whether IFI16 mediated caspase-1 activation is unique to KSHV or a common mechanism against nuclear replicating DNA viruses with episomal genomes.