Interaction of the 89K murine cytomegalovirus immediate-early protein with core histones

Abstract

The conditions that permit the interaetion of immediate-early proteins of murine cytomegalovirus (MCMV) with DNA were studied. Chromatography of extracts from infected cells on MCMV DNA cellulose and calf thymus DNA cellulose showed that pp89, the regulatory major immediate-early protein, interacts with DNA and dissociates at salt concentrations between 0.3 and 0.6 M NaCI. pp76, a cleavage product of pp89, and additional minor ie1 proteins eluted already at low ionie strength. Cellular DNA-binding factors were required for assoeiation of pp89 with DNA. These factors were identified as core histones. Chromatography of IE proteins on histone-Sepharose in the absence of DNA revealed a high-binding atfinity that was resistant to 2 M NaCI. These results suggest that pp89 has no direct DNA-binding activity. A role for an amino acid sequence homology in the N-terminal region of pp89 with histone H28 in the pp89-histone-DNA interaction is discussed.

Human Cytomegalovirus Major Immediate Early 1 Protein Targets Host Chromosomes by Docking to the Acidic Pocket on the Nucleosome Surface

“…5) tones H3-H4 (81), and several histone-associated proteins (see, e.g., references 51 and 81 to 83); however, IE2 neither associates with condensed chromatin (21,25) nor with nucleosomes (this work), at least not to the same extent as IE1. Surprisingly, even mIE1 has little affinity for nucleosomes and does not colocalize with mitotic chromatin (21,84; this work) despite reportedly binding to DNA and core histones (85,86). Accordingly, IE1 orthologs of known rodent CMVs (including mCMV and rat CMV strains) do not seem to exhibit functional CTD sequences (data not shown).…”

The Core Histone-binding Region of the Murine Cytomegalovirus 89K Immediate Early Protein

“…MCMV (mouse salivary gland virus, strain Smith, ATCC VR-194) was used to infect BALB/c mouse embryonal fibroblasts (MEFs) as described (Muench et al, 1988). Construction of vaccinia virus recombinants has been described by Volkmer et al (1987).…”

“…lmmunoprecipitation and immunofluorescence. Immunoprecipitation, gel electrophoresis and indirect immunofluorescence were carried out as described previously (Buehler et al, 1990;Muench et aL, 1988). Analysis of the cellular distribution of pp89 during mitosis was carried out on BALB/c 3T3 IE1 cells grown to 50% confluence.…”

“…Chromatography of cell extracts and pp89 fragments on histone-Sepharose was carried out as described previously (Muench et aL, 1988). Core histones H3, H2B, H2A and H4 were from Boehringer Mannheim.…”

“…The amino acid sequence of the MCMV IE1 protein contains a region of homology to histone H2B and a highly acidic carboxy-terminal region. In vitro studies have revealed that pp89 is eluted from chromatin at low salt concentration, but binds strongly to isolated histones (Muench et al, 1988). As the HCMV IE1 protein, which also contains an acidic carboxy-terminal region, recently has been shown to interact with metaphase chromosomes (LaFemina et al, 1989), we investigated whether the high affinity of pp89 for histones observed in vitro correlates with an association of pp89 with chromatin in vivo.…”