A Protein (ORF2) Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Interacts with DNA

摘要

Bovine herpesvirus 1 (BHV-1), like other members of the Alphaherpesvirinae subfamily, establishes latency in sensory neurons. The virally encoded latency-related RNA (LR-RNA) is expressed abundantly in latently infected sensory neurons and encodes several proteins, including ORF2. An LR mutant virus with stop codons at the amino terminus of ORF2 does not reactivate from latency after treatment with the synthetic corticosteroid dexamethasone, in part because it induces higher levels of apoptosis during the establishment of latency. ORF2 inhibits apoptosis, interacts with three cellular transcription factors (Notch1, Notch3, and C/EBP-α), and interferes with Notch-mediated signaling. Consequently, we predict that ORF2 expression is crucial for the latency reactivation cycle in cattle. In this study, we tested whether ORF2 interacts with nucleic acids, because it contains 18% basic amino acids and localizes to the nucleus. A subset of ORF2 proteins was associated with chromatin and preferentially associated with single-stranded DNA in transfected neuroblastoma cells (Neuro-2A). Alanine substitution of serine, threonine, and tyrosine residues in ORF2 increased the steady-state protein levels in Neuro-2A cells, and this protein preferentially interacted with double-stranded DNA. Certain in-frame transposon insertion mutants did not interact with DNA as efficiently as wild-type (wt) ORF2 did. ORF2 purified from bacteria under denaturing conditions preferentially interacted with double-stranded DNA, suggesting that the interaction between ORF2 and DNA was direct. In contrast, ORF2 purified under native conditions preferentially interacted with single-stranded DNA. We suggest that interactions between ORF2 and DNA mediate certain aspects of the latency reactivation cycle.