Studies on the in vivo methylation of replicating herpes simplex virus type 1 DNA

Abstract

Intracellular replicating DNA molecules ( g9 CsC1 = 1.725 g/cm 3) of herpes simplex virus type 1 (HSV-1) were found to be transiently methylated. Primary rabbit kidney cells infected with HSV-1 (KOS strain) were labeled with l-[ methyl- 3H]methionine at different times during the virus growth cycle. Viral DNA was deproteinized, separated from cellular DNA by centrifugation in CsCl density gradients, and treated with NaOH to avoid any RNA contamination. Analysis of this intracellular viral DNA indicated that it was maximally methylated during 4–9 hr postinfection and the methylation of viral DNA started to decrease during later stages of infection. DNA from mature virions was not found to be methylated. The methylated base was identified as 5-methylcytosine. Nicotinamide, a potent acceptor of methyl groups, effectively inhibited the production of infectious virus particles at a concentration of 50 mM. These results suggest that methylation of HSV-1 DNA during active viral DNA synthesis is a prerequisite for infectious virus production.