Identification of the bovine herpesvirus 1 circ protein, a myristylated and virion-associated polypeptide which is not essential for virus replication in cell culture.

We have recently reported immediate-early (IE) transcription over covalently joined genome ends of bovine herpesvirus 1 (BHV-1). A spliced 1.5-kb IE RNA (IER1.5) is coterminal with an unspliced 1.1-kb late RNA (LR1.1) which is transcribed from the left end of the genome. Sequence analysis reveals an open reading frame common to IER1.5 and LR1.1 predicted to encode the 247-amino-acid circ polypeptide. This paper reports on the identification of circ as a protein. Using a rabbit antiserum raised against a synthetic oligopeptide representing the carboxy terminus of the predicted circ polypeptide for Western blot (immunoblot) analyses and immunofluorescence assays, we identified a 34-kDa virion-associated protein which accumulated in the cytoplasm of infected cells. To confirm that LR1.1 indeed encoded the 34-kDa polypeptide, we inserted a DNA fragment containing circ coding sequences into the Autographa californica baculovirus genome. A group of recombinant polypeptides with sizes of 32, 34, and 35 kDa were identified by their reactivity with the antipeptide serum. Chicken egg yolk antibodies raised against total proteins of insect cells infected with the recombinant baculovirus identified the 34-kDa circ protein specified by BHV-1. The recombinant circ polypeptides and the circ protein specified by BHV-1 were both myristylated, as determined by radiolabeling with [3H]myristic acid. It was noted that the circ gene could be deleted from the BHV-1 genome without impairing virus replication in cell culture.