Autoregulation of Adenovirus Type 5 Early Gene Expression III. Transcription Studies in Isolated Nuclei

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Abstract
The rate of adenovirus RNA synthesis was compared in nuclei isolated from [human oral carcinoma KB] cells infected at 40.5.degree. C in the presence of 1-.beta.-D-arabinofuranosylcytosine with adenovirus 5 or an early temperature-sensitive mutant of adenovirus type 5, H5ts125 (ts125). In nuclei isolated at various times after infection, the maximum amount of virus RNA synthesis occurred at 6 h after infection, after which time virus RNA synthesis declined in nuclei from wild-type infections but remained high in nuclei from ts125 infections. At 12 h after infection, the amount of virus RNA synthesis was 8- to 11-fold higher in nuclei from ts125 infections than in nuclei from wild-type infections. The kinetics of virus RNA synthesis in nuclei isolated from both infections were similar. When a ts125-infected culture was shifted to 32.degree. C for 3 h (12-15 h after infection) before nucleus isolation, the amount of virus RNA synthesis in the isolated nuclei was reduced to nearly wild-type levels. A pulse-chase experiment showed little difference in degradation rates of virus RNA in isolated nuclei from wild-type and ts125 infections. Hybridization of RNA synthesized in vitro to restriction fragments of adenovirus type 5 DNA was consistent with early virus RNA. Apparently the 72,000-dalton DNA-binding protein encoded by the mutant gene in ts125 can regulate early adenovirus gene expression by inhibiting initiation of transcription of the adenovirus genome.