|Liu, Yilin - University Of Nebraska|
|Hancock, Morgan - University Of Nebraska|
|Doster, Alan - University Of Nebraska|
|Jones, Clinton - University Of Nebraska|
Submitted to: Journal of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/30/2015
Publication Date: 1/6/2016
Citation: Liu, Y., Hancock, M., Workman, A.M., Doster, A., Jones, C. 2016. ß-catenin, a transcription factor activated by canonical Wnt signaling, is expressed in sensory neurons of calves latently infected with bovine herpesvirus 1. Journal of Virology. 90(6):3148-3159.
Interpretive Summary: A major site for life-long latent infections of a-herpesvirinae subfamily members is terminally differentiated neurons. Consequently, these viruses must promote neuronal survival and maintain normal neuronal functions. We are using bovine herpesvirus 1 (BoHV-1) to study these complicated virus host interactions because the natural host can be used, corticosteroids consistently induce reactivation from latency, and BoHV-1 is a significant bovine pathogen. Like many a-herpesvirinae subfamily members, BoHV-1 expresses an abundant transcript during latency, the latency related (LR)-RNA. LR-RNA encodes two micro-RNAs and at least one protein (ORF2). In this study, we demonstrate that BoHV-1 stabilizes expression of 2 cellular transcription factors, ß-catenin and mastermind like 1 (MAML1), that are components of the Wnt signaling pathway. Wnt signaling, due to ß-catenin activation, inhibits neuro-degeneration but maintains neuronal differentiation. When reactivation from latency is initiated by the synthetic corticosteroid dexamethasone, two Wnt antagonists are induced and ß-catenin expression decreases in neurons. ORF2 stabilizes ß-catenin and MAML1 protein expression, enhances cell survival following growth factor withdrawal, and stimulates ß-catenin dependent transcription. In summary, these studies suggest ORF2 maintains neuronal survival and differentiation by stabilizing ß-catenin protein levels in latently infected neurons.
Technical Abstract: Like many a-herpesvirinae subfamily members, bovine herpes virus 1 (BoHV-1) expresses an abundant transcript in latently infected sensory neurons: the latency-related (LR) RNA. LR-RNA encodes a protein (ORF2) that inhibits apoptosis, interacts with Notch family members, interferes with Notch mediated transcription, and stimulates neurite formation in cells expressing Notch. A LR mutant virus containing stop codons at the amino-terminus of ORF2 does not reactivate from latency or replicate efficiently in certain tissues indicating LR gene products mediate crucial steps during the latency-reactivation cycle. In this study, we identified two proteins that are crucial components of the canonical Wnt signaling, ß-catenin and mastermind like 1 (MAML1) in trigeminal ganglionic neurons of latently infected, but not mock-infected calves. Most ORF2 positive neurons also express ß-catenin suggesting that ORF2 regulates ß-catenin expression. During dexamethasone-induced reactivation from latency, the mRNA expression levels of two Wnt antagonists, dickkopf1 (DKK1) and secreted frizzled protein 2 (sFRP2), were induced in bovine TG and concurrently ß-catenin protein expression was not detected in sections of TG neurons prepared from these calves. ORF2 and MAML1 stimulated ß-catenin dependent transcription in mouse neuroblastoma cells more effectively than MAML1or ORF2 alone, in part because they stabilize expression of each other. Neuroblastoma cells expressing ORF2, MAML1, and ß-catenin were highly resistant to cell death following serum withdrawal; whereas, most cells transfected with only one of these genes died. The Wnt signaling pathway interferes with neuro-degeneration but promotes neuronal differentiation. Based on these results, we propose that stabilization of ß-catenin expression by ORF2 maintains neuronal survival and differentiation.