Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25.
Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis 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 phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment.
Battista
M C
MC
Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, St. Orsola Hospital, Bologna, Italy.
Bergamini
G
G
Boccuni
M C
MC
Campanini
F
F
Ripalti
A
A
Landini
M P
MP
D000818:Animals; D019556:COS Cells; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D000939:Epitopes; D015870:Gene Expression; D006801:Humans; D051379:Mice; D009842:Oligopeptides; D010455:Peptides; D011387:Prokaryotic Cells; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C087088:UL25 protein, Cytomegalovirus; D014764:Viral Proteins; C085706:FLAG peptide