Intranuclear accumulation of subgenomic noninfectious human cytomegalovirus DNA in infected cells in the presence of ganciclovir

Abstract

In preparation for an attempt to elucidate some aspects of the interaction between ganciclovir and human cytomegalovirus (HCMV) DNA replication in cells infected with HCMV, we developed a dot blot DNA-DNA hybridization technique to quantify intracellular HCMV DNA replication. We studied the effect of ganciclovir on the time course of HCMV DNA replication in human fibroblasts. Ganciclovir resulted in complete cessation of the production of infectious virus, as detected by the plaque assay. However, viral DNA synthesis, as measured by dot blot DNA-DNA hybridization with cloned HCMV DNA BamHI C fragment probe, continued in the presence of ganciclovir at 10 times the 50% effective dose (i.e., 10 micrograms/ml). The continuation of viral DNA synthesis in ganciclovir-treated cultures leads to the intranuclear accumulation of short (subgenomic) HCMV DNA fragments. These DNA fragments are neither packaged nor released into the culture medium. Furthermore, the short DNA fragments were detected only by the BamHI C probe from the center of the unique long segment of the HCMV genome. The failure of the DNA probes from the termini of HCMV genome (BamHI-Q and HindIII-M) to detect the short DNA fragments and the intranuclear localization of these fragments suggest that these short fragments may lack the signal sequences necessary for packaging and release as infectious virions. These data strongly suggest that the anti-HCMV activity of ganciclovir is due mainly to the prevention of viral DNA chain elongation which results in the intranuclear accumulation of incomplete noninfectious viral DNA fragments.