Protection from Interferon-Induced Apoptosis by Epstein-Barr Virus Small RNAs Is Not Mediated by Inhibition of PKR

Abstract

The Epstein-Barr virus (EBV) EBER transcripts are small, highly structured RNAs able to bind to and inhibit activation of the double-stranded RNA-dependent protein kinase PKR in cell-free systems, and within latently infected B-cell lines they inhibit alpha interferon-induced apoptosis that is believed to be mediated through PKR. Here, we address the consequences of EBER expression for PKR activation in vivo in response to alpha interferon. In agreement with published findings, either EBV infection or the EBERs alone protected Burkitt lymphoma cells from alpha-interferon-induced apoptosis. However, utilizing multiple phosphorylation state-specific antibodies to monitor PKR activation within cells in response to interferon, we demonstrate that the EBERs are unable to inhibit phosphorylation of either cytoplasmic or nuclear PKR. Concordantly, a direct substrate of PKR, the ␣ subunit of eukaryotic initiation factor 2 (eIF-2␣), was equally phosphorylated in EBV-positive and EBV-negative cells following interferon treatment. Therefore, EBER inhibition of alphainterferon-induced apoptosis, and potentially other PKR-mediated events, is unlikely to be mediated through direct inhibition of PKR, as previously thought.

Sensing of latent EBV infection through exosomal transfer of 5′pppRNA

“…Indeed, chronic activation of antiviral immunity by erroneous detection of extracellular RNA drives autoimmunity in predisposed individuals (20). Although binding of proinflammatory EBERs to cytoplasmic sensors has been reported in vitro (9)(10)(11)(12), there is little evidence that this may occur in latently infected B cells in a physiologically relevant context (21), but there are multiple indications that EBERs are secreted from infected cells (9,22,23).…”

“…Both double-stranded loops and the uncapped 5′-triphosphate terminus of EBERs serve as ligands for pathogen recognition receptors (PRRs) (9)(10)(11)(12). Because infected individuals (estimated at 90% of the world population) generally have no symptoms, it seems that EBER production in healthy individuals elude cell-intrinsic danger signaling.…”

Pathologic Significance of EBV Encoded RNA in NPC

“…To support this hypothesis, researchers have made it clear that when EBV-negative Akata cells transfected with EBERs were analysed, PKR autophosphorylation in vitro was inhibited (Nanbo et al, 2002). However, Ruf reported that EBERs did posses a modest ability to protect the cell against interferon-induced apoptosis, but this process was independent of PKR-eIF-2α activation (Ruf et al, 2005). Thus Swaminathan suggested that EBERs might inhibit apoptosis while it was unlikely that inhibition of PKR was the primary mechanism for this effect (Swaminathan, 2010).…”

“…Subsequent studies revealed that EBERs were responsible for these phenotypes (Komano et al, 1999;Ruf et al, 2005). Transfection of the EBER genes into EBV-negative Akata clones restored the capacity for growth in soft agar, tumorigenicity in SCID mice, resistance to apoptotic inducers, and upregulated expression of bcl-2 that was originally retained in parental EBV-positive Akata cells but lost in EBV-negative subclones.…”

Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

“…[33][34][35] EBER RNAs can also inhibit apoptosis induced by various signals including IFN-a, but it remains controversial as to whether this antiapoptotic activity is mediated through the inhibition of PKR. [36][37][38] To address 1). In the second experiment, we assessed the activation of eukaryotic initiation factor 2a (eIF2a) using the pATF4-UTR-Luc reporter construct.…”