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

Abstract

The rate of adenovirus RNA synthesis was compared in nuclei isolated from cells infected at 40.5°C in the presence of 1-β- d -arabinofuranosylcytosine with adenovirus 5 or an early temperature-sensitive mutant of adenovirus type 5, H5 ts 125 ( ts 125). 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 ts 125 infections. At 12 h after infection, the amount of virus RNA synthesis was 8- to 11-fold higher in nuclei from ts 125 infections than in nuclei from wild-type infections. However, the kinetics of virus RNA synthesis in nuclei isolated from both infections were similar. When a ts 125-infected culture was shifted to 32°C for 3 h (12 to 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 ts 125 infections. Hybridization of RNA synthesized in vitro to restriction fragments of adenovirus type 5 DNA was consistent with early virus RNA. These results support the idea that the 72,000-dalton DNA-binding protein encoded by the mutant gene in ts 125 can regulate early adenovirus gene expression by inhibiting initiation of transcription of the adenovirus genome.