@article{mbs:/content/journal/jgv/10.1099/0022-1317-64-2-373, author = "LaFemina, Robert L. and Hayward, Gary S.", title = "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", journal= "Journal of General Virology", year = "1983", volume = "64", number = "2", pages = "373-389", doi = "https://doi.org/10.1099/0022-1317-64-2-373", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-64-2-373", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", keywords = "replication", keywords = "immediate-early protein", keywords = "hybridization", keywords = "phosphonoacetate", abstract = "SUMMARY 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.", }