Relative abundances of early virus RNA species in the cytoplasm of cells infected with wild-type adenovirus type 5 (WT Ad5) and a temperature-sensitive "early" mutant, H5tsl25 (ts125), were compared by hybridization kinetics using separated strands of HindIII restriction endonuclease fragments of Ad5 DNA. 1-f8-D-Arabinofuranosylcytosine (ara-C) was used to limit transcription to early virus genes in cells infected by WT virus. At 40.5°C, a restrictive temperature for ts125, three to seven times as much virus RNA from all four early regions of the genome accumulated in the cytoplasm of cells infected by the mutant as accumulated in cells infected by WT. At 32°C, no such difference in the relative abundances of cytoplasmic virus RNA was observed. The capacity to synthesize a 72,000-dalton (72K) virus polypeptide, presumably the single-stranded DNAbinding protein that is defective in ts125 at restrictive temperatures, was compared in cells infected at 40.5°C in the presence of ara-C with the mutant or WT Ad5. The rate of 72K polypeptide synthesis, measured by sodium dodecyl sulfatepolyacrylamide gradient gel electrophoresis of [35S]methionine-labeled polypeptides and autoradiography, was greater at 15 h after infection in ts125-infected cells than in cells infected by WT. A time course experiment showed that the rate of synthesis of the 72K polypeptide increased continuously in ts125-infected cells during the first 15 h of infection, relative to the rate in WT-infected cells. These data are consistent with the hypothesis that Ad5 early gene expression is modulated by the product of an early gene, the 72K DNA-binding protein.

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