We prepared a murine monoclonal antibody reactive to a human cytomegalovirus (HCMV)-induced nuclear protein with an Mr of 68000. Expression of the 68K protein was compared with the major immediate early (IE) 72K protein in various cell types after infection with HCMV or microinjection of plasmid DNA containing the major IE gene. The 68K nuclear protein was detected 2 to 3 h after appearance of the 72K protein in human embryonal lung (HEL) cells infected with HCMV. The 68K protein was distributed throughout the cytoplasm in the late phase of infection, while the 72K protein remained chiefly in the nucleus. The 68K protein was barely detected in the cells under IE conditions by immunoprecipitation, but, together with the 72K protein, it was expressed after microinjection of cloned DNA, containing only the major IE region (region 1), into the nuclei of HEL cells. The 72K protein was expressed in nuclei 2 h after microinjection, whereas the 68K protein was detected 4 to 5 h after the injection. The 68K protein was expressed after microinjection in non-permissive rodent fibroblasts or non-permissive transformed human cells in which these proteins were not expressed after viral infection.Immunoprecipitations after chase-labelling from IE conditions or after partial digestions suggested that the 68K protein is neither a degradation nor a modification product of the major IE 72K protein.
SUMMARYIn a previous study we showed that cells infected with guinea-pig cytomegalovirus (GPCMV) contain large amounts of a non-structural 50K nuclear protein that is detectable by immunoelectron microscopy using monoclonal antibodies. The present study shows that this 50K protein is a DNA-binding protein, as determined by singlestranded DNA affinity chromatography, and a phosphorylated protein as demonstrated by immunoprecipitation using [32P]orthophosphate_labelled cells. This protein binds both viral and host cellular double-stranded and single-stranded DNA, assayed by a simple method using DNA linked to a nylon membrane. Induction of the 50K protein in GPCMV-infected cells was highly dependent on viral DNA synthesis, which was detected by dot hybridization using a cloned GPCMV DNA probe. Synthesis of the 50K protein was significantly impaired when phosphonoacetic acid was added to the culture medium. Induction of the 50K protein was detected about 6 h before the appearance of the 76K viral matrix protein.
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