Growth in Agarose of Human Cells Infected With Cytomegalovirus

Abstract

Cultured cells transformed by certain DNA and RNA viurses develop the ability to multiply when suspended in a medium gelled with agarose. Normal fibroblasts obtained from human diploid lines do not grow in agarose medium but will do so after being transformed by Rous sarcoma virus. We report here that after infection by human cytomegalovirus (CMV) human diploid fibroblasts acquire the capacity to grow in agarose medium for several generations. Clones of infected cells grew for weeks although in every case they ultimately underwent lysis, apparently related to persistent replication of virus. Virus was inoculated at very low multiplicities (high dilution) in an effort to select non-infectious defective virions still capable of inducing cell stimulation. Viral suspensions were also irradiated (UV-CMV) to preferentially suppress infectivity. Dilute or irradiated virus yielded similar colonies of replicating cells although permanent transformation was not achieved. One clone derived from UV-CMV infected cells was passaged four times before undergoing lysis. During these passages the cells exhibited alterations in morphology and orientation. Our inability to obtain permanently transformed cells after inoculation with irradiated or diluted CMV may be due to complementation or the failure of the CMV DNA to achieve integration with the cellular genome. If permanent cellular transformation by human CMV is achieved in subsequent experiments, it will still remain to demonstrate whether this phenomenon bears any relationship to neoplasia in man. It is of interest that CMV is taxonomically related to EB and Herpes simplex type 2 viruses which have been associated with human cancer.