HEp-2 cells were infected with herpes simplex virus type 1 and extracted with 0.25% Triton X-100 and 0.1 M NaCl. The extract was sedimented on sucrose gradients, and the fractions containing the endogenous DNA polymerizing activity (replication complex) were collected. The properties of the replication complex were partially characterized. Under optimal conditions 375 pmol of dTMP per micrograms of DNA was incorporated, which corresponds to about 50% replication of preexisting viral DNA. The replication complex was shown to contain only DNA of viral origin by its density in CsCl. By using specific assays for DNA polymerases alpha, beta, gamma, and herpes simplex virus, we found that only the viral DNA polymerase copurified with the replication complex.
Herpes simplex virus DNA synthesis in a partially purified soluble extract from infected cells
PIGNATTI, Pierfranco;BERTAZZONI, Umberto
1979-01-01
Abstract
HEp-2 cells were infected with herpes simplex virus type 1 and extracted with 0.25% Triton X-100 and 0.1 M NaCl. The extract was sedimented on sucrose gradients, and the fractions containing the endogenous DNA polymerizing activity (replication complex) were collected. The properties of the replication complex were partially characterized. Under optimal conditions 375 pmol of dTMP per micrograms of DNA was incorporated, which corresponds to about 50% replication of preexisting viral DNA. The replication complex was shown to contain only DNA of viral origin by its density in CsCl. By using specific assays for DNA polymerases alpha, beta, gamma, and herpes simplex virus, we found that only the viral DNA polymerase copurified with the replication complex.
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