Replicative forms of human cytomegalovirus DNA with joined termini are found in permissively infected human cells but not in non-permissive Balb/c-3T3 mouse cells

Balb/c-3T3 mouse cells were found to be highly restricted non-permissive hosts for human cytomegalovirus (HCMV) strain Towne. These cells did not produce infectious progeny virions, did not permit virus DNA replication, and allowed expression of only a single, major, virus-specific, immediate-early polypeptide. Virus DNA synthesis was examined by three different experimental approaches. In infected Balb/c-3T3 cells, no ³²P-labelled newly synthesized DNA was found at the virus density in CsCl gradients and no virus-specific fragments were detected after cleavage with restriction enzymes. Similarly, hybridization experiments revealed no net increase in total virus DNA over the amount of input virus-specific DNA sequences. In contrast, infected permissive human fibroblast cells synthesized ³²P-labelled virus-specific DNA fragments and accumulated greatly increased amounts of total hybridizing virus DNA. Experiments with a cloned BamHI L-S joint fragment probe provided evidence for the formation of either circular or concatemeric replicative forms of HCMV DNA in which all half-molar terminal fragments were missing and the proportion of quarter-molar joint fragments increased. These forms were abundant in the first 48 h after infection of permissive human cells and mature linear monomeric forms accumulated thereafter. No detectable joining of the termini of input virus DNA occurred in either non-permissive Balb/c-3T3 cells or in human fibroblast cells in the presence of phosphonoacetic acid. In the infected Balb/c-3T3 cells a single major protein corresponding to the 68K immediate-early polypeptide could be detected within 2 h after cycloheximide reversal. Few, if any, other virus proteins were synthesized at later times or in the absence of inhibitors. The 68K protein was overproduced in Balb/c-3T3 cells to such an extent that it became a major component of the nuclear fraction and could be readily detected by direct staining procedures in polyacrylamide gels.