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-,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

Adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2

“…Regardless of the precise nature of its involvement, our results with Ad5tsl25 and Ad5tslO7 suggest that the early region 2 of Ad is important for AAV growth. It is of interest that our results with Ad5tsl49, which confirm those of others (24, 51), indicate that the Ad early region 5 protein, which is altered by this mutation and is required for Ad DNA synthesis, is not required for AAV growth. Recently, with Ad5 mutants having deletions in early region 1 (31), we have also shown that the expression of both complementation groups of this region (i.e., region la and lb) are apparently required for AAV DNA replication and viral growth (Laughlin et al, Abstr.…”

“…With the mutant helpers, AAV DNA synthesis was decreased fourto fivefold, which does not directly account for the much greater de-crease in accumulation of AAV particles. The E72 protein is required for Ad DNA replication (54) and is also involved in the negative regulation of its own, as well as other, Ad early mRNA's (5,11). It is not clear from the present results if the decrease in AAV DNA synthesis with the Ad early region 2 mutants reflects a direct effect on AAV DNA synthesis or AAV protein synthesis.…”

“…The RNAs a, c, e, and g have a spliced-out region with approximate map coordinates of 41/48. It is not yet known whether the four different 5' termini at map positions 5,13,19, and 39/40 reflect differences in promoter (lanes b and c), AAVplus Ad5tsl25 (lanes d and e), or uninfected cells (lanes f and g). Immunoprecipitation was performed with normal rabbit serum (lanes b, d, and t) or anti-AAV2 immunoglobulin G (lanes c, e, and g).…”

Different biological activities of the hetero- and homodimers formed by the 47- and 43-kilodalton proteins of transcription factor ATF/E4TF3

“…Transcription from the E4 promoter is also regulated by viral trans-acting factors. The adenovirus ElA gene products stimulate transcription (1,16), and the E2A gene products, viral DNA-binding proteins, specifically inhibit it (2,11,23).…”

Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit

“…However, the ts125 mutation is subject to frequent reversion (37a), and the mutated DNA-binding protein is partially functional at 37ЊC (10). Furthermore, E4 mRNAs are significantly increased in H5ts125-infected cells at the nonpermissive temperature (1). Upregulation of E4, a natural target of DNAbinding protein repression (23), is therefore possible after in vivo transfer of second-generation adenovectors, at least in those cells which exhibit E1A-substituting activities.…”