U(s)9, a stable lysine-less herpes simplex virus 1 protein, is ubiquitinated before packaging into virions and associates with proteasomes

The U(s)9 gene of herpes simplex virus 1 encodes a virion tegument protein with a predicted M-r of 10,000. Earlier studies have shown that the gene is not essential for viral replication in cells in culture. We report that (i) U(s)9 forms in denaturing polyacrylamide gels multiple overlapping bands ranging in M-r from 12,000 to 25,000; (ii) the protein recovered from infected cells or purified virions reacts with anti-ubiquitin antibodies; (iii) autoradiographic images of U(s)9 protein immunoprecipitated from cells infected with [S-35] methionine-labeled virus indicate that the protein is stable for at least 4 h after entry into cells (the protein was also stable for at least 4 h after a l-h labeling interval 12 h after infection); (iv) antibody to subunit 12 of proteasomes pulls down U(s)9 protein from herpes simplex virus-infected cell lysates; and (v) the U(s)9 gene is highly conserved among the members of the alpha subfamily of herpes viruses, and the U(s)9 gene product lacks lysines. We conclude that U(s)9 is a lysineless, ubiquitinated protein that interacts with the ubiquitin-dependent pathway for degradation of proteins and that this function may be initiated at the time of entry of the virus into the cell.