Breaking Human Cytomegalovirus Major Immediate-Early Gene Silence by Vasoactive Intestinal Peptide Stimulation of the Protein Kinase A-CREB-TORC2 Signaling Cascade in Human Pluripotent Embryonal NTera2 Cells

Abstract

. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.Persons with impaired cellular immunity risk tissue-invasive disease from reactivation of latent human cytomegalovirus (HCMV). HCMV reactivation in tissues or blood of immunocompetent patients suffering illness from another cause also occurs but usually goes unnoticed and is self-limiting. For instance, nearly one-third of patients who are critically ill or in septic shock have detectable findings of HCMV reactivation in their bloodstream (12,26,43,45,80). HCMV reactivation infection in intestinal tissues with preexisting inflammatory disease (e.g., inflammatory bowel disease) is also well described for immunocompetent patients (28,39,40,51). The precise triggering mechanisms that underlie HCMV reactivation are unknown.So far, only cells of myeloid lineage have been determined to fulfill criteria for cellular sites of HCMV latency in vivo. In healthy HCMV-seropositive persons, precursors of macrophages and dendritic cells, including CD34ϩ hematopoietic progenitor cells, carry latent HCMV genomes at a low frequency (75). The terminal differentiation of these cells into a macrophage-or dendritic cell-like phenotype is a prerequisite for HCMV reactivation ex vivo (67, 76). However, this reactivation appears to be a rare event, suggesting that other, as yet unidentified factors may promote HCMV reactivation. Stimulation with tumor necrosis factor alpha (TNF-␣) or gamma interferon may promote HCMV reactivation from differentiated counterparts of monocytic-dendritic cell precursors that had been infected latently in vitro or in vivo (25,66,67,76).For both HCMV and murine CMV, viral major immediateearly (MIE) gene expression is greatly restricted or shut off during viral latency, and the productive viral life cycle cannot advance without this expression (57,74,75,78). The MIE enhancer/promoter controlling expression is regulated by the coordinated actions of multiple types of cis-acting elements (57). These elements are bound by cellular transcription factors whose functions are modulated by input supplied by the cell, the virus, and the external surrounding (57). Higher-order chromatin structure contributes to this regulation. Heterochromatin components amass on the inactive MIE enhancer/promoter in viral latency, whereas the chromatin signatures of transcriptional activity predominate at the active MIE enhancer/ promoter in acute and reactivation infections (47,67). MIE enhancer/promoter silencing is also favored by innate antiviral mechanisms that partly involve the repressive actions of nuclear ND10 domain components (e.g., hDAXX, PML, ATRX, and histone deacetylase [HDAC]) (52) but does not involve CpG methylation of the MIE enhancer/promoter (29).Quiescent HCMV infection of human NTera2/D1 cells (NT2 cells) is a tractable model system for studying the regulation of HCMV MIE enhancer/promoter reactivation (37, 54). NT2 cells share many phenotypic features with pluripotent

Phorbol Ester-Induced Human Cytomegalovirus Major Immediate-Early (MIE) Enhancer Activation through PKC-Delta, CREB, and NF-κB Desilences MIE Gene Expression in Quiescently Infected Human Pluripotent NTera2 Cells

“…Ancillary data coming from recent work sparked an awareness of phorbol ester's ability to also increase HCMV MIE gene expression levels in quiescently infected NT2 cells (59). This invited speculation that MIE gene activation might be achieved as well through a separate regulatory pathway that involves protein kinase C (PKC) (59).…”

“…In a similar NT2 cell model, the inhibition of HDAC by TSA was found to disrupt heterochromatin nucleation at the MIE enhancer/promoter (42), akin to the chromatin disruption that accompanies HCMV reactivation in endogenously infected dendritic cells (49). We have shown recently with NT2 cells that the stimulation of the cAMP-PKA-CREB signaling cascade by either vasoactive intestinal peptide (VIP) or forskolin (FSK) activates the HCMV MIE enhancer/promoter via cAMP response elements (CREs) to relieve HCMV genome silence (25,59). The transcriptional coactivator transducer of regulated CREB-2 (TORC2) is a critical part of this signaling pathway and must also be activated by VIP or FSK via a PKA-dependent mechanism in order to desilence the MIE genes (59).…”

“…In infected NT2 cells, either the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (36) or the stimulation of cyclic AMP (cAMP)-dependent protein kinase A (PKA) activity (25,59) immediately turns on the transcription of HCMV MIE genes in previously dormant HCMV genomes (25,59). This rapidly induced transcriptional activation is not the result of cellular differentiation (42,59).…”

“…This rapidly induced transcriptional activation is not the result of cellular differentiation (42,59). Conversely, RA fails to reverse the MIE enhancer/promoter silence in quiescently infected NT2 cells (36), but its use to first convert these cells into differentiated NT2 (NT2-D) cells subsequently enables the activation of the HCMV MIE enhancer/promoter immediately upon viral infection (16).…”

“…We have shown recently with NT2 cells that the stimulation of the cAMP-PKA-CREB signaling cascade by either vasoactive intestinal peptide (VIP) or forskolin (FSK) activates the HCMV MIE enhancer/promoter via cAMP response elements (CREs) to relieve HCMV genome silence (25,59). The transcriptional coactivator transducer of regulated CREB-2 (TORC2) is a critical part of this signaling pathway and must also be activated by VIP or FSK via a PKA-dependent mechanism in order to desilence the MIE genes (59).…”