Localization of Sequences in a Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 That Inhibits Apoptosis and Interferes with Notch1-Mediated trans -Activation of the bICP0 Promoter

Abstract

Bovine herpesvirus 1 (BHV-1) infection induces clinical symptoms in the upper respiratory tract, inhibits immune responses, and can result in life-threatening secondary bacterial infections. Following acute infection, BHV-1 establishes latency in sensory neurons within trigeminal ganglia. Periodically, reactivation from latency occurs, resulting in virus transmission. The latency-related (LR) RNA is abundantly expressed in latently infected sensory neurons, suggesting that LR gene products regulate the latency-reactivation cycle. An LR mutant virus with stop codons at the amino terminus of the first open reading frame (ORF) in the LR gene (ORF2) does not reactivate from latency, in part because it induces higher levels of apoptosis in infected neurons. ORF2 inhibits apoptosis in transiently transfected cells, suggesting that it plays an important role in the latency-reactivation cycle. ORF2 also interacts with Notch1 or Notch3 and consequently inhibits their ability to trans-activate the bICP0 early and glycoprotein C promoters. In this study, we identified ORF2 sequences that were necessary for inhibiting cold shock-induced apoptosis or Notch1-mediated trans-activation of the bICP0 early promoter and stimulation of productive infection. Relative to ORF2 sequences necessary for inhibiting apoptosis, distinct domains in ORF2 were important for interfering with Notch1-mediated transactivation. Five consensus protein kinase A and/or protein kinase C phosphorylation sites within ORF2 regulate the steady-state levels of ORF2 in transfected cells. A nuclear localization signal in ORF2 was necessary for inhibiting Notch1-mediated trans-activation but not apoptosis. In summary, ORF2 has more than one functional domain that regulates its stability and functional properties.

Potential Role for a β-Catenin Coactivator (High-Mobility Group AT–Hook 1 Protein) during the Latency-Reactivation Cycle of Bovine Herpesvirus 1

“…4B). As previously reported (6,7,28), ORF2 was primarily localized at the rim of the nucleus (Fig. 4C).…”

“…ORF2 contains a nuclear localization signal (NLS) (Fig. 8A) that, when deleted (the ORF2ΔNLS mutant), expresses a protein that localizes to the plasma membrane (28). The ORF2ΔNLS mutant was not able to enhance ␤-catenindependent transcription more effectively than the empty vector.…”

A Bovine Herpesvirus 1 Protein Expressed in Latently Infected Neurons (ORF2) Promotes Neurite Sprouting in the Presence of Activated Notch1 or Notch3

“…To induce neurite sprouting, 24 h after transfection, cells were seeded into new plates at a low density of 2,000/cm 2 and starved in medium with 0.5% serum for 3 days. Cells were then fixed and stained, and a ␤-galactosidase (␤-Gal) assay was performed as previously described (44) Dual-luciferase reporter assay. Neuro-2A cells (8 ϫ 10 5 ) were seeded into 60-mm dishes containing EMEM with 10% FCS at 24 h prior to transfection.…”

“…ORF2 stably interacts with Notch3 or Notch1, and these interactions impair the ability of Notch to stimulate viral gene expression and productive infection (26,44). Although these studies indicated that ORF2 interferes with the ability of Notch family members to stimulate viral promoters and productive infection, they did not address whether ORF2 influences activated Notch in the context of known cellular functions.…”

“…A significant difference between the four transposon mutant constructs and WT ORF2 was observed (P Ͻ 0.05). Since ORF2-95, unlike the other transposon mutant constructs, expresses lower protein levels in Neuro-2A cells than WT ORF2 does (44), it is difficult to distinguish whether its impaired function was due to protein expression levels or if an essential inhibitory domain was disrupted. The remainder of the transposon mutant constructs restored neurite formation similar to that seen with WT ORF2, regardless of whether they were cotransfected with Notch1 or Notch3 (Fig.…”