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Ten temperature-sensitive (ts) mutants of adenovirus type 12 which produce plaques at 31 but not at 38.5 C have been isolated after mutagenesis with nitrosoguanidine or nitrous acid. The mutants have been classified into six separate complementation groups. DNA-DNA hybridizations have shown that at 38.5 C the ts 401 and 406 mutants of groups B and E, respectively, synthesized less than 10% of the normal level of viral DNA. The two mutants were also defective in the production of late proteins at the nonpermissive temperature, as shown by fluorescent-antibody tests and analysis by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Genetic recombination between the ts viruses 401 and 406 has been demonstrated; the recombination frequency for the wild-type virus production was 17.7%. Both mutants induced an increase in thymidine kinase activity at 38.5 C. Moreover, the two viral DNA-defective mutants shut off host DNA synthesis at the restrictive temperature. It is striking that at 38.5 C ts virus 401 transformed two to eight times more hamster cells than the wild-type virus, whereas ts virus 406 transformed at a frequency similar to the wild-type virus.

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of 3 H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. 3 H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much 3 H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.

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