Differentiation-Coupled Induction of Human Cytomegalovirus Replication by Union of the Major Enhancer Retinoic Acid, Cyclic AMP, and NF-κB Response Elements

Abstract

Triggers and regulatory pathways that effectively link human cytomegalovirus (HCMV) major immediate early (MIE) latent-lytic switch activation with progeny production are incompletely understood. In the quiescently infected human NTera2 cell model of primitive neural stem cells, we found that costimulation with vasoactive intestinal peptide (V) and phorbol ester (P) synergistically activated viral infection, but this effect waned over time. Coupling retinoic acid (R), an inducer of neuronal differentiation, to VP pulse stimulation attenuated the decline in viral activity and promoted the spread of the active infection through concentric layers of neighboring cells as cellular differentiation progressed. R stimulation alone was unable to activate the infection. The MIE enhancer cis-regulatory mechanisms responsible for this result were characterized by a strategy of combinatorial mutagenesis of five cis-acting element types (retinoic acid receptor binding elements [RARE], cyclic AMP [cAMP] response elements [CRE], NF-κB binding sites [kB], serum response element, and ETS/ELK-1 binding site) and multiple methods of assessment. We found that the CRE and kB combination sets the preinduction enhancer tone, is the major initiator and amplifier of RVP-induced MIE gene expression, and cooperates with RARE during cellular differentiation to enhance viral spread. In predifferentiated NTera2, we also found that the CRE-kB combination functions as initiator and amplifier of unstimulated HCMV MIE gene expression and cooperatively interacts with RARE to enhance viral spread. We conclude that RVP-stimulated signaling cascades and cellular differentiation operate through the enhancer CRE-kB-RARE core in strengthening induction of HCMV MIE gene expression in linkage with viral propagation.IMPORTANCE Cytomegalovirus-seropositive persons commonly lack detectable levels of cytomegalovirus replication, even when profoundly immunocompromised. In a human NTera2 cell model of primitive neural stem cells carrying resting cytomegalovirus genomes, we show that costimulation of protein kinase A and C-delta signaling cascades in conjunction with retinoic acid-induced neuronal differentiation brings about progeny virus propagation. Iterated DNA binding sites for retinoic acid receptor, CREB, and NF-κB family members in the cytomegalovirus major enhancer are at the crux in the pathway to HCMV activation. The stimulated CREB and NF-κB binding site combination vigorously initiates and amplifies the active cytomegalovirus infection and cooperates with activated retinoic acid receptor binding sites to further promote viral proliferation and spread between differentiated cells. These results support a paradigm in which a specific combination of stimuli coupled with cellular differentiation satisfies a core cis-activating code that unlocks enhancer silence to repower the cycle of cytomegalovirus propagation.

Human cytomegalovirus IE2 drives transcription initiation from a select subset of late infection viral promoters by host RNA polymerase II

“…Viral stocks were serially diluted to attain an MOI of 0.5-0.8 infectious units per HFF cell as determined by immunofluorescence assay of IE1/IE2 protein-positive cells at 24 h pi, using murine monoclonal antibody MAB810 (EMD Millipore, 1:1000 dilution) followed by a secondary goat antimouse IgG (H+L) antibody conjugated to Alexa Fluor 555 (Thermo Fisher Scientific, A-21422). The results informed the calculation of the number of infectious units per mL of viral inoculum [63,64].…”

“…TRIzol Reagent was used to isolate whole-cell RNA according to the manufacturer's instructions (Invitrogen, 15596026). Reverse transcription (RT) and quantitative real-time PCR (qPCR) were performed using methods described previously [63] and PCR primers listed in S2 Table. The IE1, IE2, MIE, and UL128 primer sets span the introns for their respective genes. All RT-qPCR analyses were performed in triplicate (triplicate infections, triplicate treatment conditions) in 12-well plates and results normalized to host GAPDH RNA.…”

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells

“…For example, deletion of the CREB response elements from the proximal canonical MIEP resulted in a defect in HCMV reactivation in DCs whereas a HCMV virus with NF-kB site MIEP deletions was less affected [16]. However, in other models of reactivation from latency, a clear phenotype with NF-kB MIEP deletions has been observed [41][42][43]. Most recently, blockade of AP1 activity has also been demonstrated to impact on HCMV MIEP-driven gene expression in both the Kasumi 3 and a CD34+model of latency and reactivation [13].…”

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…These pathways include mitogen-activated kinase (MAPK) signalling both via extracellular signal-regulated kinase (ERK) 1 and 2 including RAF1 (MAPKKK upstream of ERK) as well as via p38 MAPK [168][169][170][171][172]. Other kinases thought to be involved in the IE phase of HCMV infection include adenosine monophosphate-activated protein kinase (AMPK) [173], hematopoietic cell kinase (a src family kinase) [174], cyclin-dependent kinases (CDKs) [175], protein kinase A [176] and mitogen and stress activated kinase (MSK) [128]. The activation of kinase signalling pathways in the initial infection phase comes with multiple, mostly beneficial consequences for the virus including major IE gene activation.…”