Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes.
Stable DNA-transfected Vero cell lines that express the major immediate-early nuclear antigen (IE68) of HCMV-(Towne) have been established. Immunofluorescence staining with monoclonal antibodies revealed that the protein was distributed either in a uniform diffuse nuclear pattern or as punctate nuclear granules in up to 80% of the cells in these cultures. In addition, 1 to 2% of the positive nuclei gave a distinctive staining pattern suggesting an association with the chromosomes of mitotic cells. Colcemid-blocking studies confirmed that most of the IE antigen was localized in the vicinity of condensed chromosomes in all metaphase cells after methanol fixation. In contrast, the SV40 large T-antigen protein was found to be preferentially excluded from metaphase chromosomes in a similar colcemid-treated human cell line. In transient expression assays, 1 to 2% of IE antigen-positive Vero, 293, or Balb/c3T3 cells also displayed a metaphase chromosome association pattern. Mapping studies using deletion and truncation mutants revealed that the monoclonal antibodies recognized epitopes encoded within the small NH2-terminal exons that are common to both the IE1 and IE2 gene products. However, an intact exon-4 (IE1) region, but not the exon-5 (IE2) region of the HCMV IE gene complex, was required for conferring both the normal diffuse nuclear localization pattern and the chromosome-association properties. Furthermore, removal of the glutamic acid-rich COOH-terminal coding portions of exon-4 resulted in aberrant staining patterns with production of large, phase-dense nuclear globules in all positive cells. An association between the IE68 IE1 protein and metaphase chromosomes was also detected after HCMV-(Towne) infection in a small proportion of both nonpermissive Balb/c3T3 cells and permissive HF cells. We conclude that the IE1 acidic nuclear phosphoprotein displays some properties similar to those of the EBNA-1 protein of Epstein-Barr virus and suggest that it may potentially play a role in maintenance of the latent state of HCMV DNA.
Lafemina
R L
RL
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Pizzorno
M C
MC
Mosca
J D
JD
Hayward
G S
GS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D002875:Chromosomes / Q000032:analysis*; D003587:Cytomegalovirus / Q000235:genetics* / Q000276:immunology; D000939:Epitopes / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006801:Humans; D017874:Immediate-Early Proteins; D008677:Metaphase; D010957:Plasmids; D014162:Transfection; D014709:Vero Cells
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D017874:Immediate-Early Proteins; C044582:immediate-early proteins, cytomegalovirus