DNA labeled during phosphonoacetate inhibition and following its reversal in herpesvirus infected cells

Summary

Human embryonic lung cells were pre-equilibrated with phosphonoacetate and³²P orthophosphate label, then infected with phosphonoacetate-sensitive herpes simplex virus (HSV) type 1. Analyses of viral DNA produced in these cells showed the following. i) Viral DNA was synthesized in infected cells exposed to 100 µg of the drug per ml of medium but not in cells exposed to four-fold higher concentrations of the drug. ii) At 300 µg/ml a region of the DNA between 0.58 and 0.69 map units became transiently labeled, but the restriction endonuclease fragment containing these sequences migrated more slowly than the corresponding fragment from virion DNA. iii) Viral DNA extracted from infected cells 1.5 hours post drug withdrawal (300 µg/ml) was preferentially labeled in 2 regions of the genome mapping between 0.17 and 0.23 and 0.58–0.69 map units. This finding is in agreement with a report ofFriedman et al. (8) suggesting that HSV DNA contains two different sites of initiation. In addition a 4.8×10⁶ molecular weight fragment was also preferentially labeled. This fragment could represent a smaller, aberrantly migrating fragment from the 0.17–0.27 map unit region of the DNA. (iv) Viral DNA extracted from infected cells at longer intervals after drug withdrawal showed an increasing gradient of radioactivity progressively labeling the genome. These results are consistent with the hypothesis that viral DNA has at least two sites of initiation of DNA synthesis and that both sites are within the L component of the DNA. Alternatively, the results could be interpreted as two sites of localized synthesis (repair) that are detected at high concentrations of phosphonoacetate and immediately following reversal of inhibition of DNA synthesis. The results do not exclude the possibility that secondary sites in both L and S are utilized late in infection or in untreated cells.