Retrograde Regulation by the Viral Protein Kinase Epigenetically Sustains the Epstein-Barr Virus Latency-to-Lytic Switch To Augment Virus Production

Abstract

Herpesviruses are ubiquitous, and infection by some, like Epstein-Barr virus (EBV), is nearly universal. To persist, EBV must periodically switch from a latent to a replicative/lytic phase. This productive phase is responsible for most herpesvirus-associated diseases. EBV encodes a latency-to-lytic switch protein which, upon activation, sets off a vectorially constrained cascade of gene expression that results in production of infectious virus. While triggering expression of the switch protein ZEBRA is essential to lytic cycle entry, sustaining its expression is equally important to avoid premature termination of the lytic cascade. We report that the viral protein kinase (vPK), encoded by a gene that is kinetically downstream of the lytic switch, sustains expression of ZEBRA, amplifies the lytic cascade, increasing virus production, and, importantly, prevents the abortive lytic cycle. We find that vPK, through a noncanonical site phosphorylation, activates the cellular phosphatidylinositol 3-kinase-related kinase ATM to cause phosphorylation of the heterochromatin enforcer KAP1/TRIM28 even in the absence of EBV genomes or other EBV proteins. Phosphorylation of KAP1 renders it unable to restrain ZEBRA, thereby further derepressing and sustaining its expression to culminate in virus production. This partnership with a host kinase and a transcriptional corepressor enables retrograde regulation by vPK of ZEBRA, an observation that is counter to the unidirectional regulation of gene expression reminiscent of most DNA viruses. IMPORTANCE Herpesviruses infect nearly all humans and persist quiescently for the life of the host. These viruses intermittently activate into the lytic phase to produce infectious virus, thereby causing disease. To ensure that lytic activation is not prematurely terminated, expression of the virally encoded lytic switch protein needs to be sustained. In studying Epstein-Barr virus, one of the most prevalent human herpesviruses that also causes cancer, we have discovered that a viral kinase activated by the viral lytic switch protein partners with a cellular kinase to deactivate a silencer of the lytic switch protein, thereby providing a positive feedback loop to ensure successful completion of the viral productive phase. Our findings highlight key nodes of interaction between the host and virus that could be exploited to treat lytic phase-associated diseases by terminating the lytic phase or kill cancer cells harboring herpesviruses by accelerating the completion of the lytic cascade.

A promiscuous inflammasome sparks replication of a common tumor virus

“…Specifically, the TXNIP−NLRP3 inflammasome depletes KAP1, a barrier to EBV lytic activation. While we have previously shown that KAP1 restricts expression of multiple lytic genes including BZLF1, and is deactivated via phosphorylation after successful entry into the lytic phase leading to amplification of the lytic cascade (15,17), our present findings demonstrate that depletion instead of phosphorylation of KAP1 triggers entry into the lytic phase through derepression of ZEBRA, the EBV replication switch. This drop in KAP1 level, together with elevation in TXNIP, serves as a prelytic marker to identify cells in which EBV is poised to enter the lytic cycle.…”

“…relies on the activities of the viral protein kinase which phosphorylates the cellular ATM kinase at S2996; activated ATM then phosphorylates KAP1 at S824 to derepress multiple lytic genes (15)(16)(17). Of note is that, although KAP1 depletion was linked to caspase-1 in another study, depletion was observed late in the lytic cascade coincident with activation of apoptotic pathways, and the mechanism of caspase-1 activation was unexplored (22).…”

“…Cellular stressors such as hyperglycemia and ROS are known to activate the NLRP3 inflammasome through interactions between NLRP3 and the cellular arrestin protein TXNIP (14). In earlier work, we have demonstrated that the master epigenome regulator KAP1/TRIM28 serves as a barrier to EBV lytic cycle entry by enforcing heterochromatin at EBV lytic genes including BZLF1 (15)(16)(17). Our experiments now show that lytic triggers cause assembly of the TXNIP−NLRP3 inflammasome to activate procaspase-1; active caspase-1 then causes partial loss of KAP1/TRIM28 to turn the expression of the viral replication switch protein on within a subpopulation of cells, thereby disrupting quiescence in those cells.…”

Histone Loaders CAF1 and HIRA Restrict Epstein-Barr Virus B-Cell Lytic Reactivation

“…EBV late genes are subsequently induced and include factors required for virion assembly and spread ( 10 ). Retrograde signals support ongoing lytic replication through subversion of chromatin-based repressors ( 18 ).…”

A heterochromatin inducing protein differentially recognizes self versus foreign genomes

“…Recent findings show that in a defense strategy, TRIM28/KRAB-ZFP complexes also epigenetically silence foreign genomes and in the process, regulate the life cycle of certain viruses. These include extrachromosomal genomes of persistent viruses such as Epstein-Barr virus (EBV), Kaposi's Sarcoma-Associated virus (KSHV), and human cytomegalovirus (CMV) [9][10][11][12][13][14][15][16]. Specifically for EBV and KSHV, we have previously shown that recruitment of TRIM28 to multiple viral lytic genes silences the destructive lytic program, thereby allowing these viruses to maintain latency.…”