An adenovirus type 5 early gene function regulates expression of other early viral genes.

Abstract

We have identified an adenovirus type 5 (Ad5) early gene function located in early region 1 which is require for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4. Mutant d1312 (lacks the segment between 1.5 and 4. Recently, we isolated a group of deletion mutants that lack portions of early region 1 (unpublished data). These mutants were isolated by selecting viral DNAs that lack the Xba I restriction endonuclease cleavage site-at 4 map units by the procedure of Jones and Shenk (7). These mutants are propagated in 293 cells (an Ad5-transformed human embryonic kidney cell line; ref. 8); they do not replicate in HeLa cells.In this report we show that the deletions in these mutants make predictable alterations in the cytoplasmic mRNAs encoded by early region 1. Further, we find that one of these mutants, d1312, defines a region 1 gene function that is required for the production of early cytoplasmic mRNAs corresponding to early regions 2, 3, and 4.MATERIALS AND METHODS Cells and Viruses. The 293 cells were provided by F. Graham and have been described (8 11g. RNA was isolated from cells (treated with 20,ug of cytosine arabinoside per ml) 8 hr after infection at a multiplicity of 50 plaque-forming units (PFU)/cell. Infected cells were divided into nuclear and cytoplasmic fractions. Cells were suspended in isotonic buffer (10 mM Tris1HCl, pH 7.8/1.5 mM MgCl2/150 mM NaCI), and Nonidet P-40 was added to 0.6% after the mixture was cooled to 40C. The mixture was held on ice for 10 mi. then mixed on a Vortex for 10 sec; the nuclei were pelleted by centrifugation. The supernatant was the cytoplasmic fraction and the pellet was the nuclear fraction after two additional washes in isotonic buffer.We prepared nuclear RNA by dissolving the nuclear pellet in a low pH sodium dodecyl sulfate buffer (50 mM NaOAc, pH 5.2/100 mM NaCI/10 mM EDTA/0.5% sodium dodecyl sulfate), extracting the RNA twice at room temperature with an equal volume of phenol (equilibrated with 50 mM NaOAc, pH 5.2), extracting it once with chloroform/isoamyl alcohol, 49:1 (vol/vol), and precipitating the RNA by addition of 2 vol of ethanol. The RNA was resuspended in 10 mM Tris-HCl, pH 7.4/10 mM MgCI2, electrophoretically purified DNase I was added to 50 ,ug/ml, and the solution was incubated at 37°C for 30 min. Finally, the RNA was reextracted with phenol and with chloroform/isoamyl alcohol, and precipitated with ethanol. Cytoplasmic RNA was prepared by mixing the cytoplasmic cellular fraction with 3 vol of 100 mM Tris-HCl (pH 9), extracting twice at room temperature with phenol (pH 9)/chloroform/isoamyl alcohol, 500:100:1 (vol/vol)

Selective induction of human heat shock gene transcription by the adenovirus E1A gene products, including the 12S E1A product.

“…The adenovirus ElA gene is a regulatory gene that specifies products that both activate (2,16,35) and repress (3,11,50) transcription of unlinked genes. During a lytic viral infection, five early transcription units are stimulated by the action of the product of the 13S ElA RNA, a 289-amino-acid protein (42).…”

Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products.

“…The best characterized is the ElA-dependent trans activation of the remainder of the adenovirus early genes during productive infection in permissive human cells (3,27,35,47,69). The products of the ElA region alone are also sufficient to establish primary rodent cells (25,54), and in addition, the ElA products can act in concert with the adenovirus E1B region (11,16,28,40,58,66) or with other oncogenes such as the ras gene product (54) to fully transform various rodent cells.…”

Presence of the adenovirus E1A-like activity in preimplantation stage mouse embryos.

“…The E1A-dependent E2A gene (9) is transcribed when EC stem cells are infected with dl312 (7). However, the E2A gene is not activated when infected into differentiated derivatives of EC stem cells (7).…”