THE UL3 OPEN READING FRAME OF HERPES-SIMPLEX VIRUS TYPE-1 CODES FOR APHOSPHOPROTEIN

Based on sequence analysis, the protein encoded by the UL3 open readin g frame (ORF) of herpes simplex virus type 1 (HSV-l) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encod ing the complete sequence of the UL3 ORF in a baculovirus expression s ystem. Western blotting was done using polyclonal antibody raised agai nst synthetic UL3 peptides. Two major baculovirus-UL3 expressed protei n bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 k Da were detected. None of the expressed UL3 protein species were susce ptible to tunicamycin treatment, suggesting that they were not N-linke d glycosylated. Cell fractionation studies indicated that the UL3 prot ein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylati on. In contrast, the baculovirus expressed UL3 protein was phosphoryla ted as judged by P-32(i)-labeling. Immunoprecipitation followed by SDS -PAGE demonstrated a single P-32(i)-labeled UL3 related band with an a pparent molecular weight of 33 kDa, indicating that the UL3 protein wa s a phosphoprotein. Antibodies produced in mice vaccinated with baculo virus-UL3 protein reacted with two UL3 related HSV-I bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3.