Expression of the acidic nuclear immediate-early protein (IE1) of human cytomegalovirus in stable cell lines and its preferential association with metaphase chromosomes

Robert L. Lafemina, Marie C. Pizzorno, Joseph D. Mosca, Gary S. Hayward

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)584-600
Number of pages17
JournalVirology
Volume172
Issue number2
DOIs
StatePublished - Oct 1989

ASJC Scopus subject areas

  • Virology

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