Location: Animal Health Genomics
Project Number: 3040-32000-036-002-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2021
End Date: Jun 30, 2025
Objective:
Bovine herpesvirus 1 (BoHV-1) infects cattle causing respiratory and genital diseases. Following acute infection in mucosal linings of the eye, nose, or mouth, BoHV-1 establish a lifelong latent infection (a kind of dormancy) in sensory neurons within trigeminal ganglia (TG). Stress triggers BoHV-1 reactivation from latency which facilitates spread of the virus to other cattle and increases the incidence of bovine respiratory disease (BRD). A single intravenous injection of the synthetic corticosteroid dexamethasone (DEX) to latently infected calves always induces reactivation from latency and virus shedding occurs. DEX, via activation of the glucocorticoid receptor (GR), stimulates key viral promoters and productive viral infection. Interestingly, genes associated with the canonical Wnt/B-catenin signaling pathway are differentially expressed in TG of latently infected calves relative to TG from uninfected calves and latently infected calves treated with DEX. Further analysis of RNA-sequencing (RNAseq) data revealed the phosphatidylinositol-3-Kinase (PI3K)/Akt/mTOR signaling axis, axonal guidance, neuronal survival, and cAMP response element-binding (CREB) protein signaling pathways are more active during latency. Exciting unpublished studies in this proposal demonstrate Akt1 and Akt2 plus a LR non-coding RNA impair GR-mediated transcriptional activation. Based on these observations, we hypothesize LR gene products promote the maintenance of latency by interfering with stress-induced transcription and co-opting key cellular signaling pathways that sustain normal neuronal functions and ensure neuronal survival. Studies in the three objectives of this grant application will test this hypothesis.
Objective 1: Characterize cellular signaling pathways differentially expressed during latency.
Objective 2: Analyze LR gene products and cellular factors that impair stress-induced reactivation.
Objective 3: Characterize viral and cellular factors that promote neurogenesis.
Approach:
Objective 1: Immuno-histochemistry and RT-PCR studies will confirm key signaling pathways discussed above are differentially expressed during latency. RNAseq studies will also compare expression of these signaling pathways in calves latently infected with a LR mutant virus that has impaired establishment of latency and does not reactivate efficiently.
Objective 2: Studies in this objective will identify LR RNA sequences important for repressing stress (GR) induced transcription. Neuro-2A cells (mouse neuroblastoma cells) and FBBC-1 cells (SV40 LTA immortalized bovine brain cells) will be transfected with ORF-2 RNA sequences along with a stress responsive promoter construct containing the luciferase reporter gene. Cell cultures will be treated with DEX, which mimics the effects of stress, and expression of the reporter gene will be measured in the presence of various ORF-2 RNAs to localize the sequences that interfere with stress-mediated transcription. Additional studies will test whether other LR gene products (micro-RNAs, ORF-E, ORF-1) have similar functions and if ORF2 RNA sequences cooperate with Akt1 and and Ak2 to impair stress induced transcription following treatment with DEX.
Objective 3: We will test whether LR gene sequences cooperate with Akt family members to regulate stress induced neuronal differentiation in vitro. Utilization of specific Akt inhibitors will confirm Akt protein kinase activity is important for GR-mediated transactivation and neurite sprouting, an assay that measures neurogenesis. Finally, we will perform IHC to test whether certain differentially expressed genes during latency, are induced during neurite sprouting.
