Human Cytomegalovirus I. Purification and Characterization of Viral DNA

Abstract

Two human strains (AD-169 and C87) and one simian strain (GR2757) of cytomegalovirus (CMV) have been purified from the extracellular fluids of virus-infected cultures by sedimentation through a sucrose gradient followed by brief centrifugation in a preformed gradient of CsCl. Enveloped virus particles were located in the density region, 1.219 g/cm 3 , and nucleocapsids at 1.263 g/cm 3 . Purified viral DNA, both human and simian, sedimented in the region of 55 S in neutral sucrose gradients with herpes simplex type I DNA as a marker; the molecular weight of the CMV DNA was estimated as approximately 10 8 . The density of the viral DNA determined by analytical ultracentrifugation was 1.716 g/cm 3 for the human strains and 1.710 g/cm 3 for the simian strain. Tritiated viral complementary RNA synthesized in vitro with Escherichia coli transcriptase has been used for detection and localization of viral genome in membrane hybridization and in situ cytohybridization. Newly synthesized viral DNA appeared 24 h after infection and localized at two acrocentric areas; later the viral DNA distributed in a band resembling intranuclear inclusions 48 to 70 h after infection. Total DNA synthesis began to increase 24 h after infection and reached its peak at 70 h; RNA synthesis increased at 13 h, and reached its peak at 24 to 33 h. The viral DNA was also labeled with 3 H-TTP by repair-synthesis in vitro with Kornberg's enzyme in order to analyze the purity of the DNA and for detection of viral DNA by DNA-DNA reassociation kinetics.