Epstein-Barr virus is a lymphotropic herpesvirus that establishes a lifelong infection in its host once it is acquired. EBV has been associated with several cancers, such as Burkitt’s lymphoma (BL), Hodgkin’s lymphoma, and post-transplant lymphoproliferative disease. This oncogenic potential has been attributed to EBV due to its ubiquitous presence in different tumor types and also for its ability to infect and transform human B cells in vitro resulting in an immortalized lymphoblastoid cell line [1, 2]. Like all herpesviruses, EBV has both a lytic and a latent phase; however, EBV is unique amongst other herpesviruses as it has three latencies (Lat. I, II, and III), each defined by a tightly regulated subset of genes [3]. During initial infection, EBV replicates in the oral epithelial and/or local mucosal-associated lymphoid tissue [4, 5], with over 80 proteins expressed during production of infectious virions [6]. This is referred to as the lytic or productive phase. The temporal cascade of gene expression that follows can be subdivided into immediate early (IE), early (E), late (L), and latent (Lt). Initially, the IE gene products are expressed and act as transactivator proteins required for the expression of the E genes [7]. These E genes assist in viral DNA replication, including transcription and translation of the lytic transcripts (L) encoding the viral structural proteins and Lt. genes required for persistence of the EBV genome [7]. At the culmination of EBV replication, the virus particles are packaged and released. After primary infection of epithelial cells, EBV enters B cells where latency is established and the EBV genome is maintained as extrachromosomal episomes in the host cell. Approximately one in a million B cells at any given time are latently infected with EBV, and can periodically reactivate enabling the spread and survival of the virus [8].

All EBV-associated cancers identified to date occur as a result of the virus’ latent infection. In most cases, EBV infection is asymptomatic; whereas immunosuppressed individuals may develop EBV-associated lymphoid or epithelial-derived carcinomas. During latency, EBV can evade the host immune system, regulate apoptosis and promote B cell development and survival [9, 10]. Despite evidence that host CD8⁺CTL responses keep latently infected B cells under control, even healthy individuals experience reactivation and shedding of EBV [11, 12]. To this day, host factors important for controlling the lytic to latent or latent to lytic switch of EBV are not well understood.

Interferon-γ-inducible protein 16 (IFI16) is a multifunctional and ubiquitous host protein, initially found to be constitutively expressed in human lymphoid cells [13]. Since then, IFI16 has been reported to have critical roles in antiviral immunity. Kerur, et al. first published that IFI16 acts as a nuclear innate DNA sensor upon primary infection of human dermal microvascular endothelial cells to detect the KSHV genome in the nucleus, resulting in inflammasome activation [14]. This was the first report to demonstrate that inflammasome activation can occur in the nucleus, while identifying IFI16 as the key nuclear pathogen sensor. Additional findings showed that IFI16 constitutively senses both the latent genomes of KSHV and EBV (latencies I, II, and III) to induce the inflammasome [15, 16]. Specifically, IFI16 combines with adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) and effector protein procaspase-1 to activate the IFI16/ASC/procaspase-1 inflammasome following the detection of dsDNA viruses [14]. This response leads to maturation and cleavage of the proinflammatory cytokines interleukin 1β (IL-1), IL-18 and IL-33 to the infected area, ultimately helping to fight infection. Inflammasome activation occurs in the cytoplasm, requiring IFI16 acetylation and translocation from the nucleus of the infected cell to the cytoplasm [16].

IFI16 also has important roles as a viral restriction factor. For example, Lo Cigno et al. found that overexpression of IFI16 using transfection of the adenovirus-based vector AdVIFI16 severely impaired human papilloma virus 18 replication in both NIKS and U2OS cells, while reducing viral transcription [17]. Similar studies by the same group revealed that siRNA-mediated knockdown of IFI16 resulted in enhanced human cytomegalovirus (HCMV; a β-herpesvirus) replication, and overexpression in the same system yielded decreased viral load and HCMV DNA copy number [18]. Moreover, it was reported that HSV-1 has reduced viral titers/DNA replication and gene expression in the presence of IFI16, which is in part due to IFI16’s chromatin remodeling abilities [19].

Recent studies on KSHV biology expanded our understanding of IFI16’s role in antiviral immunity as it was shown to be crucial for maintaining KSHV latency. Upon lytic cycle induction with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in the latently KSHV-infected B-cell lymphoma BC-3 and BCBL-1 cell lines, IFI16 is targeted for proteasomal degradation [20]. This is mediated in part by KSHV late lytic transcripts, and degradation of IFI16 results in increased levels of KSHV lytic proteins and the overall viral genome abundance [20]. Furthermore, Ansari et al. showed by fluorescence in situ hybridization that IFI16 colocalizes with the EBV genome in lymphoblastoid (latency III) and Raji (latency I) cell nuclei [16]. However, despite the extensive studies performed on lytic replicating DNA viruses, recent studies on latent KSHV infections and the observation that IFI16 recognizes the EBV genome, studies on the role of IFI16 in EBV biology are lacking.

The finding that IFI16 is required for maintenance of KSHV latency [20] suggests that IFI16 may also be important for EBV latency. Here, we show for the first time that IFI16 is indeed essential for maintaining latency in the EBV positive, latently I-infected Akata and MUTU-1 cell lines. Knockdown of IFI16 by siRNA-mediated transfection in Akata cells resulted in the efficient reduction of IFI16 mRNA and protein levels, increased EBV lytic gene expression, and increased global EBV viral load. Lytic cycle induction by transforming growth factor β (TGF-β; MUTU-1) and the chemical inducer TPA (Akata) effectively reduced levels of IFI16 protein up to 80% as compared to their uninduced counterparts. This reduction in IFI16 protein correlated to elevated levels of lytic EBV transcripts, increased genome copy number, and a redistribution of IFI16 from the nucleus to the cytoplasm as well. Collectively, these studies demonstrate a vital role for IFI16 during the latent to lytic switch, and provide insight into how EBV interacts with its host.

Antiviral immunity against foreign, invading pathogens is vital for ensuring the survival of the infected host. Some viruses such as herpesviruses, however, are never cleared, but instead alternate between a lytic and latent state throughout the life of the infected individual. During lytic infection, progeny virus are produced and are recognized by the immune system, resulting in a myriad of cellular responses against the virus.

To overcome host detection, EBV has evolved a complex set of latencies unique to EBV. It’s during these latencies that EBV-associated malignancies such as BL, nasopharyngeal carcinoma, and lymphoproliferative diseases (amongst others) arise. Unfortunately, the only available treatments for such diseases include chemotherapy and immunotherapy, which, if effective, can have significant toxic side effects and leave the patients permanently deformed [32–34]. Therefore, identifying host proteins that are involved in EBV infection is crucial for developing new strategies to combat viral-induced diseases.

Besides its role as an integral part of the inflammasome during dsDNA infection, IFI16 has been reported to have diverse gene regulatory and chromatin remodeling functions during HSV-1, HCMV, HPV, and KSHV infections [17, 19, 20, 35]. IFI16 acts as a negative regulator in its ability to bind to the promoters of all temporal gene classes in HSV-1 while inhibiting transcription factor binding such as Oct1, TBP, and RNA Pol II [19]. Moreover, IFI16 restricts viral DNA replication in HPV in part by its ability to epigenetically modify viral promoters [17]. Gariano et al. showed that over-expression of IFI16 in human embryo lung fibroblasts resulted in decreased viral yield and total DNA abundance and that this was due to IFI16’s ability to bind to the HCMV promoter UL54, ultimately blocking Sp-1-like binding sites and preventing both early and late viral gene expression [18]. Additionally, silencing of IFI16 using siRNA reversed these effects, enhancing viral replication, which are similar to the results observed in our EBV experiments [18]. In the present study, siRNA-mediated KD of IFI16 in Akata cells results in lytic reactivation of EBV, as measured by increases in the viral genome and all temporal class gene expression (Fig. 1c & d). Contrarily, overexpression of IFI16 reversed these effects, leading decreased EBV replication and gene expression compared to samples treated with TPA alone (Fig. 1f & g). These results suggest that IFI16 is involved in lytic cycle suppression, and therefore, may directly contribute to disease progression as all EBV-associated malignancies occur during latency.

The activation of lytic genes was observed in both Akata cells induced with TPA and MUTU-1 cells induced with TGF-β, with Akata cells displaying statistically higher levels of induction (Fig. 2c and d vs. Fig. 4c and d). Differences in viral genome abundance, lytic gene expression, and IFI16 localization observed in our IFA studies (Figs. 3 and 5) could be due to i) the origin of the cell lines (a Japanese BL vs. a Kenyan BL), ii) the effectiveness of the inducer (TPA vs. TGF-β), iii) the ability of the cells to respond to their respective inducers over time (possible loss of sensitivity of cells to respond after extensive passages), or iv) the spontaneous loss of virus from the cells [36–38].

To date, IFI16 degradation has only been reported to occur in HSV-1 de novo infection [39–41] and lytic reactivation of KSHV-infected cells [20]. Recently, it was reported that IFI16 is polyubiquitinated and degraded via the proteasome-mediated pathway in the KSHV latently infected B lymphoma cell lines BCBL-1 and BC3 [20]. This degradation was shown to occur at least in part by late lytic transcripts, as treatment with PAA restored IFI16 protein levels to that of uninduced controls [20]. Using similar approaches with our EBV models, we did not observe any significant increase in IFI16 protein despite blocking protein degradation with MG132 (data not included) or the transcription of EBV late lytic transcripts with PAA (Fig. 6a-d). Rather, our results suggest that the changes in IFI16 protein may be due to IE or E genes, or perhaps a combination of the two. Furthermore, the fact that IFI16 mRNA abundance steadily increases from 0 to 96 h post induction with TPA (Fig. 2d) despite the reduction observed in protein levels, suggests that the reduction is occurring via a post-translational event. Nonetheless, it is clear that KSHV and EBV use distinct mechanisms in response to foreign, invading pathogens.

In these studies, we found that IFI16 protein levels are reduced during reactivation of the EBV lytic cycle. EBV is associated with >90% of endemic BL cases, lymphoproliferative diseases, and is implicated in gastric carcinoma, the fifth leading cause of cancer morbidity in the world [42]. IFI16 has previously been shown to be required for the establishment of KSHV latency and acts as a negative regulator of HSV-1 viral gene expression. Our studies, for the first time, implicate IFI16 as a requirement for the maintenance of EBV latency and suggest that IFI16 gets targeted post-translationally for degradation by a currently unknown mechanism.

The findings presented here provide evidence that EBV uses IFI16 to maintain its latent state. We show that IFI16 protein levels decrease following lytic cycle reactivation, implying an important role for IFI16 in the maintenance of latency and as a negative regulator of lytic cycle induction. Importantly, identification of IFI16 in the regulation of EBV latency will provide future insight into treating EBV-related malignancies and the cellular-induced immune control mechanisms that control them.