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SummaryBalb/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.

journal of general virology

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