DNA synthesis-dependent relief of repression of transcription from the adenovirus type 2 IVa2 promoter by a cellular protein

Self Cite

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. During the late phase of human adenovirus type 5 (Ad5) infection, cellular protein synthesis is severely inhibited, while viral late proteins are made in large quantities (3, 6). Such preferential expression of viral genes is the result of posttranscriptional regulatory mechanisms: several viral gene products, including VA-RNA1 and the L4 100-kDa protein, contribute to selective translation of viral late mRNAs (see references 18, 64, and 85 for reviews), while the E1B 55-kDa and E4 Orf 6 proteins induce selective export of these mRNAs from the nucleus (reviewed in references 25, 33, and 39). In infected cells, these last two early proteins form a complex (84) that has been implicated in regulation of mRNA export (12, 19). Indeed, the E1B 55-kDa and E4 Orf 6 proteins colocalize to specific sites within infected cell nuclei, the peripheral zones of replication centers (70). Transcription of viral late genes and at least initial processing of viral pre-mRNAs take place at these same sites (4,14,74,75). Mutations that prevent synthesis of the E4 Orf 6 protein or reduce binding of this to the E1B 55-kDa protein (83) result in both mislocalization of the E1B 55-kDa protein and defects in export of viral late mRNAs (39, 70). These properties indicate that E4 Orf 6 protein-dependent recruitment of the E1B protein to the specialized nuclear sites at which viral late pre-mRNAs are synthesized promotes selective export of the processed mRNAs. The observation that the accumulation of viral mRNAs at enlarged interchromatin granules, which form in infected cells as the late phase progresses, correlates with efficient late mRNA export (4, 13) provides further support for the view that efficient mRNA export is intimately coupled to the organization of infected cell nuclei. However, the molecular basis of such coupling remains unknown, nor has the cellular export pathway by which viral late mRNAs are transported from the nucleus to the cytoplasm been identified.AThe E1B 55-kDa protein contains a leucine-rich nuclear export signal (NES) that is recognized by the cellular exportin 1 export receptor and mediates shuttling of the viral protein when it is synthesized either alone or in Ad5-infected cells (26,58). It has also been reported that the E4 Orf 6 protein contains a similar NES nece...

Section: Discussionmentioning

confidence: 93%

Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

González

Wen-ying

Finnen

et al. 2006

Self Cite

“…Whereas early-phase gene products are primarily concerned with providing the ideal environment for viral DNA replication, the late genes encode predominantly the structural proteins that allow the assembly of the virus particle (1). Gene expression switches from early to late via a small class of intermediate genes that become activated around the time that viral DNA replication begins (2–4). These events have been most widely studied in human adenovirus type 5 (HAdV-C5 [Ad5]).…”

Section: Introductionmentioning

confidence: 99%

“…MLP activation is achieved upon the onset of viral DNA replication by the intermediate gene products IX and IVa2, the latter in conjunction with the L4 products 22K and/or 33K (7–9). IVa2 expression commences after an unknown cellular repressor bound to its promoter is titrated out by the excess of nascent viral genomes (2, 3). Upon activation of the MLP, L4-22K and L4-33K additionally contribute to the correct expression of the full repertoire of adenovirus late proteins by influencing the splicing of the MLTU pre-mRNA (10–12); L4-22K also cooperates with IVa2 to promote packaging of viral DNA into nascent capsids (7).…”

Section: Introductionmentioning

confidence: 99%

The Human Adenovirus 5 L4 Promoter Is Activated by Cellular Stress Response Protein p53

Wright

Leppard

2013

During adenovirus infection, the emphasis of gene expression switches from early genes to late genes in a highly regulated manner. Two gene products, L4-22K and L4-33K, contribute to this switch by activating the major late transcription unit (MLTU) and regulating the splicing of its transcript. L4-22K and L4-33K expression is driven initially by a recently described L4 promoter (L4P) embedded within the MLTU that is activated by early and intermediate viral factors: E1A, E4 Orf3, and IVa2. Here we show that this promoter is also significantly activated by the cellular stress response regulator, p53. Exogenous expression of p53 activated L4P in reporter assays, while depletion of endogenous p53 inhibited the induction of L4P by viral activators. Chromatin immunoprecipitation studies showed that p53 associates with L4P and that during adenovirus type 5 (Ad5) infection, this association peaks at 12 h postinfection, coinciding with the phase of the infectious cycle when L4P is active, and is then lost as MLP activation commences. p53 activation of L4P is significant during Ad5 infection, since depletion of p53 prior to infection of either immortalized or normal cells led to severely reduced late gene expression. The association of p53 with L4P is transient due to the action of products of L4P activity (L4-22K/33K), which establish a negative feedback loop that ensures the transient activity of L4P at the start of the late phase and contributes to an efficient switch from early- to late-phase virus gene expression.

“…Expression of pIX, IVa2, and late genes of Ad is detected only after Ad DNA synthesis has occurred. It has been shown that IVa2 transcription is regulated by a cellular repressor that is titrated out upon Ad genome replication (17,18), and MLP is regulated by IVa2 in conjunction with the late L4-22K and -33K proteins (2,26). Our previous study had shown that UXP expression is detected only in cells at the late stage of infection; no UXP was detected in the presence of cytosine arabinoside (an inhibitor of DNA replication).…”

Section: Discussionmentioning

confidence: 99%

“…The same phenomenon was demonstrated for the Ad IVa2 promoter. The IVa2 promoter was unable to drive efficient synthesis of GFP in the absence of a simian virus 40 (SV40) origin of replication in a reporter assay in a transient-transfection system (17). Furthermore, it has been well documented in superinfection experiments that replication of the DNA template is required for maximal activation of expression of pIX, IVa2, and late-region L2 to L5 proteins (11,24,37).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

Bai

Tollefson

Wold

2010

We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant-and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp ؊158 and ؉30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.