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.
The triggering mechanisms underlying reactivation of human cytomegalovirus (HCMV) in latently infected persons are unclear. During latency, HCMV major immediate-early (MIE) gene expression breaks silence to initiate viral reactivation. Using quiescently HCMV-infected human pluripotent embryonal NTera2 cells (NT2) to model HCMV reactivation, we show that vasoactive intestinal peptide (VIP), an immunomodulatory neuropeptide, immediately and dose-dependently (1 to 500 nM) activates HCMV MIE gene expression. This response requires the MIE enhancer cyclic AMP response elements (CRE). VIP quickly elevates CREB Ser133 and ATF-1 Ser63 phosphorylation levels, although the CREB Ser133 phosphorylation level is substantial at baseline. VIP does not change the level of HCMV genomes in nuclei, Oct4 (pluripotent cell marker), or hDaxx (cellular repressor of HCMV gene expression). VIP-activated MIE gene expression is mediated by cellular protein kinase A (PKA), CREB, and TORC2. VIP induces PKA-dependent TORC2 Ser171 dephosphorylation and nuclear entry, which likely enables MIE gene activation, as TORC2 S171A (devoid of Ser171 phosphorylation) exhibits enhanced nuclear entry and desilences the MIE genes in the absence of VIP stimulation. In conclusion, VIP stimulation of the PKA-CREB-TORC2 signaling cascade activates HCMV CRE-dependent MIE gene expression in quiescently infected NT2 cells. We speculate that neurohormonal stimulation via this signaling cascade is a possible means for reversing HCMV silence in vivo.
Yuan
Jinxiang
J
Department of Internal Medicine, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
Liu
Xiaoqiu
X
Wu
Allen W
AW
McGonagill
Patrick W
PW
Keller
Michael J
MJ
Galle
Courtney S
CS
Meier
Jeffery L
JL
D000956:Antigens, Viral / Q000378:metabolism*; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics*; D003586:Cytomegalovirus Infections / Q000378:metabolism / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology*
D000956:Antigens, Viral; C115163:CREB1 protein, human; C493245:CRTC2 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; D004279:DNA, Viral; D017874:Immediate-Early Proteins; D014157:Transcription Factors; C044582:immediate-early proteins, cytomegalovirus; D014660:Vasoactive Intestinal Peptide; D017868:Cyclic AMP-Dependent Protein Kinases