Adenovirus early region 4 is essential for normal stability of late nuclear RNAs

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216

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19-and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.

Section: Discussionmentioning

confidence: 93%

mentioning

confidence: 99%

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

Querido

Marcellus

Lai

et al. 1997

Self Cite

216

“…In cells infected with a virus that cannot express the 55-kDa E1B protein (E1B-55 kDa) cellular mRNA is exported from the nucleus at near normal rates whereas viral mRNA fails to be transported (40,53). A similar phenotype is observed in cells infected with a virus that cannot express the 34-kDa protein of E4 (E4-34 kDa) (28,60,61). Because the phenotype of viral mutants unable to express both proteins is no more severe than that of viruses unable to express either E1B-55 kDa or E4-34 kDa protein (9,14), it has been proposed that a complex composed of the E4-34 kDa protein and the E1B-55 kDa protein is responsible for usurping the control of mRNA transport during an Ad infection.…”

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confidence: 87%

“…Late viral transcripts with unused splice donor sites were more dependent on the E1B-55 kDa protein for efficient transport than fully spliced transcripts (39). The work of Ketner and associates has suggested that both the E4-34 kDa protein and the E4 ORF3 (open reading frame 3) protein enhance the stability of the viral RNA in the nucleus (8,9,60,61). Additional studies have further suggested that the ORF3 protein and the 34-kDa (ORF6) protein of E4 express partially overlapping functions with respect to viral growth (30).…”

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confidence: 99%

Adenovirus early region 4 34-kilodalton protein directs the nuclear localization of the early region 1B 55-kilodalton protein in primate cells

Goodrum

Shenk

Ornelles

1996

The localization of the adenovirus type 5 34-kDa E4 and 55-kDa E1B proteins was determined in the absence of other adenovirus proteins. When expressed by transfection in human, monkey, hamster, rat, and mouse cell lines, the E1B protein was predominantly cytoplasmic and typically was excluded from the nucleus. When expressed by transfection, the E4 protein accumulated in the nucleus. Strikingly, when coexpressed by transfection in human, monkey, or baby hamster kidney cells, the E1B protein colocalized in the nucleus with the E4 protein. A complex of the E4 and E1B proteins was identified by coimmunoprecipitation in transfected HeLa cells. By contrast to the interaction observed in primate and baby hamster kidney cells, the E4 protein failed to direct the E1B protein to the nucleus in rat and mouse cell lines as well as CHO and V79 hamster cell lines. This failure of the E4 protein to direct the nuclear localization of the E1B protein in REF-52 rat cells was overcome by fusion with HeLa cells. Within 4 h of heterokaryon formation and with protein synthesis inhibited, a portion of the E4 protein present in the REF-52 nuclei migrated to the HeLa nuclei.Simultaneously, the previously cytoplasmic E1B protein colocalized with the E4 protein in both human and rat cell nuclei. These results suggest that a primate cell-specific factor mediates the functional interaction of the E1B and E4 proteins of adenovirus. RESULTSThe E4-34 kDa protein increases the amount of E1B-55 kDa protein seen diffusely distributed through the nucleus during an Ad infection. Previous work suggested that the E4-34 kDa protein directs the E1B-55 kDa protein to the sites of viral DNA synthesis and viral RNA processing in the nucleus during an infection with wild-type Ad (51). Although quantitative differences in protein distribution are difficult to determine by indirect immunofluorescence, the results shown in Fig. 1A suggest that the E4-34 kDa protein also increases the amount 6324 GOODRUM ET AL.

“…A similar cellular transforming or oncogenic function has also been linked to E4-ORF1 (19)(20)(21)(22)(46)(47)(48). E4-ORF6 and E4-ORF3 have also been shown to be involved in altering mRNA expression at a posttranscriptional level (39)(40)(41)(42)(43)(44). In addition, E4 products are involved in controlling the cellular transcription factor E2F (38), in E1A-induced p53-independent apoptosis (34), in modulating the phosphorylation status of cellular and viral proteins (28,35) and have recently been shown to alter the nuclear transport of other proteins (17).…”

mentioning

confidence: 95%

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo

Brough

Hsu

Kulesa

et al. 1997

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo.Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1 ؊ E4 ؊ ) vector genome cleared with a similar kinetic profile as the GV10 (E1 ؊ ) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing ␤-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.