Structure-function Analysis of a Protein Encoded by the Bhv-1 Latency Related Gene

Bovine herpes virus 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 latencyreactivation cycle. A LR mutant virus with stop codons at the amino-terminus of the first 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 trans-activation. 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.