Abstract
BackgroundEpstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It’s during the latent phase of EBV that all EBV-associated cancers, including Burkitt’s lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease arise. Interferon-γ-inducible protein 16 (IFI16) is a well-established innate immune sensor and viral transcriptional regulator involved in response to invading DNA viruses. During latency, IFI16 remains in the nucleus, in part bound to the EBV genome; however, neither its role in EBV lytic cycle or latency has been established.MethodsShort interfering RNA against IFI16 and IFI16 overexpression were used to identify the role of IFI16 in the maintenance of EBV latency I. We also studied how induction of the lytic cycle affected IFI16 using the EBV positive, latently infected Akata or MUTU-1 cell lines. Akata cells were induced with TPA and MUTU-1 cells with TGF-β up to 96 h and changes in IFI16 protein were analyzed by Western blotting and immunofluorescence microscopy. To assess the mechanism of IFI16 decrease, EBV DNA replication and late lytic transcripts were blocked using the viral DNA polymerase inhibitor phosphonoacetic acid.ResultsKnockdown of IFI16 mRNA by siRNA resulted in enhanced levels of EBV lytic gene expression from all temporal gene classes, as well as an increase in the total EBV genome abundance, whereas overexpression of exogenous IFI16 reversed these effects. Furthermore, 96 h after induction of the lytic cycle with either TPA (Akata) or TGF-β (MUTU-1), IFI16 protein levels decreased up to 80% as compared to the EBV-negative cell line BJAB. Reduction in IFI16 was observed in cells expressing EBV lytic envelope glycoprotein. The decreased levels of IFI16 protein do not appear to be dependent on late lytic transcripts of EBV but suggest involvement of the immediate early, early, or a combination of both gene classes.ConclusionsReduction of IFI16 protein levels following lytic cycle induction, as well as reactivation from latency after IFI16 mRNA knockdown suggests that IFI16 is crucial for the maintenance of EBV latency. More importantly, these results identify IFI16 as a unique host factor protein involved in the EBV lifecycle, making it a potential therapeutic target to combat EBV-related malignancies.
Highlights
Epstein-Barr virus (EBV) exhibits both lytic and latent
Interferon-γ-inducible protein 16 (IFI16) knockdown in Akata cells results in the activation of EBV lytic cycle gene expression To determine the effect of IFI16 on EBV biology, we down-regulated the gene expression of IFI16 in the latently infected Akata B cells using short interferon RNA (siRNA). siIFI16 or its control were electroporated, incubated up to 48 h, analyzed for protein, DNA and RNA
At the same time that we observed a reduction in IFI16 protein and mRNA levels, there was an increase in total EBV DNA levels as seen by real-time DNA PCR (Fig. 1c), suggesting that loss of IFI16 results in activation of EBV’s lytic cycle
Summary
Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. 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 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]. 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]
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