Adenovirus type 5 early region 1b gene product is required for efficient shutoff of host protein synthesis

Abstract

To determine the role adenovirus 5 early region lb-encoded 21-and 55-kilodalton proteins play in adenovirus productive infection, mutants have been isolated which were engineered to contain small deletions or insertions at 5.8, 7.9, or 9.6 map units. By using an overlap recombination procedure involving H5dl3l4 (A3.7 to 4.6 map units) DNA cleaved at 2.6 map units with ClaI and the adenovirus 5 XhoI-C (O to 15.5 map units) fragment containing the desired mutation, viral mutants were isolated by their ability to produce plaques on KB cell line 18, which constitutively expresses only viral early region lb functions (Babiss et al., J. Virol. 46:454-465, 1983). DNA sequence analysis of the viral mutants isolated (H5dlll8, H5dlllO, H5inl27, and H5dll63) indicates that all of the viruses contain mutations which affect the 55kilodalton protein, whereas d1118 should also produce a truncated form of the 21-kilodalton protein. When analyzed for their replication characteristics in HeLa cells, all of the mutant viruses exhibited extended eclipse periods and effected yields that were reduced to 10% or less of that produced by H5sub309 (parent virus of the^Thutants which is phenotypically identical to wild-type adenovirus 5). When compared with * Corresponding author. MATERIALS AND METHODS Cells and viruses. Monolayer cultures of human KB, 293, (21), HeLa, and KB cell line 18 (a KB cell line that constitutively expresses the bacterial XGPRT gene and the Ad2 Elb gene; 2) cells were grown in Dulbecco modified Eagle medium supplemented with 10% calf serum. Suspension cultures of human KB cells were grown in modified 202

The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.

“…Ad CB is an El-deleted recombinant AdS expressing the lacZ gene (Berkner, 1992;Kozarsky et al, 1994). The EIB mutant H5 110 has only E1B 44 kD disrupted (Babiss and Ginsberg, 1984). The E2A mutant H5 802 contains a deletion in the 72-kD single-stranded DBP (Rice and Klessing, 1985).…”

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells

“…Measurement of p53 half-life. 293 cells were grown on 60-mm-diameter plates and mock infected or infected with AdLacZ virus, and at 7.5 h postinfection (hpi), the cells were labeled for 15 min with 100 Ci of [ 35 S]methionine (1,175 Ci/mmol; Dupont NEN) per plate and then incubated further for various times with ␣-MEM supplemented with 15 mg of L-methionine (ICN Biochemicals) per ml. The cells were then lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.2, containing 150 mM NaCl, 1% [vol/vol] Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 1 mM EDTA, 500 mM sodium vanadate, 50 mM NaF, 1 mM ␤-glycerol phosphate, and 100 Kallikrein international units of aprotinin) and immunoprecipitated with a mixture of Ab1801 and Ab421.…”

“…Thus, one role of E1B products in both productive infection and cell transformation appears to be to prevent untimely E1A-induced cell death, thus permitting production of high yields of progeny or survival of transformants. In productively infected human cells, the E1B 55-kDa polypeptide also interacts with the E4orf6 protein to form complexes which enhance accumulation and transport of late viral mRNAs and induce shutoff of host cell protein synthesis (1,2,8,12,21,30,43,44,50,52,63).…”

Regulation of Protein Synthesis in Virus-Infected Animal Cells

“…(6) See text. (7) Babiss and Ginsberg (1984). Vaccinia uirus: (1) Hruby and Ball (1981); Oppermann and Koch (1976).…”