Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25

Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new s tructural protein that is present in both virion and defective viral partic les (C, J, Baldick and T. Shenk J. Virol. 70:6097-6105, 1996), In the prese nt work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the U L25 product (pUL25) during HCMV replication in human fibroblasts. Furthermo re, pUL25 was transiently expressed in its native form and fused to the FLA G epitope, in COS7 and U373MG cells, in order to compare the properties of the Isolated protein and that produced during infection. Immunoblotting ana lysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylatio n, The transcriptional analysis of the UL25 open reading frame combined wit h the study of pUL25 biosynthesis showed true late kinetics for this protei n in infected human fibroblasts. By indirect immunofluorescence both recomb inant and viral pUL25 were detected exclusively in the cytoplasm of transfe cted or infected cells. Interestingly, pUL25 was shown to localize in typic al condensed structures in the perinuclear region as already observed for o ther HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed t o be a preferential site of viral particle envelopment. Our data suggest th at pUL25 is most likely a novel tegument protein and possibly plays a key r ole in the process of envelopment.