Phorbol ester-induced human cytomegalovirus major immediate-early (MIE) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells.
The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE(+)) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-kappaB (kappaB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and kappaB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-kappaB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and kappaB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-kappaB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.
Liu
Xiaoqiu
X
University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
Yuan
Jinxiang
J
Wu
Allen W
AW
McGonagill
Patrick W
PW
Galle
Courtney S
CS
Meier
Jeffery L
JL
D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D003586:Cytomegalovirus Infections / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D010703:Phorbol Esters / Q000494:pharmacology*; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects
D017362:Cyclic AMP Response Element-Binding Protein; D017874:Immediate-Early Proteins; D016328:NF-kappa B; D010703:Phorbol Esters; D051745:Protein Kinase C-delta