The kinetics of accumulation of early virus RNA in the cytoplasm of KB cells infected at 40.5°C by wild-type (WT) adenovirus type 5 and a temperaturesensitive "early" mutant, H5ts125 (tsl25), were compared by hybridization of unlabeled RNA in solution to the 3H-labeled I strand of Ad5 DNA HindIII
The rate of adenovirus RNA synthesis was compared in nuclei isolated from cells infected at 40.5°C in the presence of 1-,8-D-arabinofuranosylcytosine with adenovirus 5 or an early temperature-sensitive mutant of adenovirus type 5, H5ts125 (tsl25). 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 8to 11-fold higher in nuclei from ts125 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 ts125-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 wildtype levels. A pulse-chase experiment showed little difference in degradation rates of virus RNA in isolated nuclei from wild-type and tsl25 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 ts125 can regulate early adenovirus gene expression by inhibiting initiation of transcription of the adenovirus genome. One of the virus genes expressed early in the lytic cycle of adenovirus type 5 (Ad5) codes for a 72,000-dalton (72K) protein, which binds to single-stranded DNA (14, 23), is required for the initiation of virus DNA synthesis (12, 25), and maps at about 60 to 65 map units within the HindIII restriction endonuclease A fragment (15) (Fig. 1). H5ts125 (ts125) is a temperaturesensitive mutant of Ad5 (9) that has a lesion in the structural gene for this protein, resulting in a thermolabile 72K protein (13, 24). At a restrictive temperature, early virus RNA accumulation in the nuclei and cytoplasm of infected cells is two-to sevenfold higher in ts125 infections than in wild-type (WT) infections at 12 h after infection in the presence of a DNA synthesis inhibitor, 1-f8-D-arabinofuranosylcytosine (ara-C) (4). To analyze the mechanism of this overproduction of early virus RNA by ts125 at a restrictive temperature, we studied virus RNA synthesis in isolated nuclei from infected cells. Analysis of RNA synthesis in isolated nuclei permits a more direct measurement of the rate of transcription than in vivo labeling pro
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