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. 2013 Nov;87(21):11617-25.
doi: 10.1128/JVI.01924-13. Epub 2013 Aug 21.

The human adenovirus 5 L4 promoter is activated by cellular stress response protein p53

Jordan Wright  1 , Keith N Leppard
Affiliations

The human adenovirus 5 L4 promoter is activated by cellular stress response protein p53

Jordan Wright et al. J Virol. 2013 Nov.

Abstract

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.

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Figures

Fig 1
Fig 1
The L4 promoter is activated by p53. (A) Schematic showing the L4 promoter reporter constructs used in this study. Plasmids were constructed using L4P DNA at the indicated positions from wild-type Ad5 genome pTG-Ad3602 subcloned into the promoterless luciferase reporter plasmid pGL-3-Basic. (B) 293 cells were transfected either with pGL3-Basic or with full-length L4P alongside expression plasmids for the proteins indicated below the graph. All transfections included a standard amount of pCMV-βgal and, total DNA input was kept constant by addition of empty vector pCI-neo as required. Cells were harvested 24 h later, and reporter gene activity was assayed. Data are presented as relative luciferase activity (RLA), normalized to the pGL-3-Basic control RLA, which was set at 1. Error bars indicate the standard deviations for three replicate samples, and data are representative of a minimum of three independent repeat experiments. (C) As for panel B, except that the core L4P reporter (positions 26018 to 26098) was used.
Fig 2
Fig 2
p53 binds with the core L4 promoter (positions 26018 to 26098). (A) 293 cells were transfected with pGL-3-Basic (no promoter), core L4P, or ΔCore L4P reporter plasmids alongside pCI-neo (−) or a cocktail of Orf3, IVa2, and p53 expression plasmids, and extracts were assayed for reporter gene activity. Other details are as in Fig. 1B. Error bars indicate the standard deviations for three replicate samples, and data are representative of a minimum of two experiments. (B) 293 cells were transfected with the L4P reporter plasmids indicated. Twenty-four hours later, cells were fixed and processed for ChIP analysis to determine p53 binding, expressed as the percentage of L4P DNA present in the lysate (see Materials and Methods).
Fig 3
Fig 3
p53 can regulate the L4 promoter in U2OS cells. (A) U2OS cells were transfected with pGL-3-Basic (no promoter) or full-length L4P together with plasmids expressing the proteins indicated, and lysates were analyzed for reporter gene activity (RLA). Other details are as in Fig. 1B. Error bars indicate the standard deviations for three replicate samples. (B) U2OS cells were transfected with control siRNA or p53 siRNA. Forty-eight hours later, cells were transfected with pCI-neo empty vector (EV) or linear pTG-Ad3602. Cells were harvested after a further 24 h and lysates analyzed for reporter plasmid activity (RLA). Error bars show the standard deviations for three replicates, and data are representative of a minimum of three independent experiments. (Inset) One replicate well from the cultures used for reporter analyses was harvested for protein analysis by Western blotting with p53 and β-actin antibodies. (C) Average RLA of nine repeat readings from three independent repeat experiments whose results are shown in panel B. Data are expressed relative to the values obtained from siControl/pTG-Ad-transfected cells. Error bars indicate the standard deviations for the nine replicates, and values were subjected to Student's t test (2-tailed, unequal variance) *, P < 0.0001.
Fig 4
Fig 4
Endogenous p53 associates with the L4 promoter during Ad5 infection. (A) HeLa cells were infected at a multiplicity of infection (MOI) of 10 FFU/cell with wild-type Ad5 and then harvested at the time points indicated. Samples were then processed for ChIP analysis to determine specific p53 binding to L4P. The amount of L4P DNA recovered from anti-p53 samples is expressed relative to that detected in control antibody immunoprecipitates. Error bars indicate the standard deviations for three replicates, and the data are representative of two experiments. (B) Cells infected as described for panel A were harvested and analyzed by Western blotting for expression of Ad early and late proteins. The migration positions of protein molecular mass markers are indicated on the left, in kDa.
Fig 5
Fig 5
p53 is required for efficient Ad5 late gene expression. (A) HeLa cells were transfected with control or p53 siRNAs. Twenty-four hours later, cells were trypsinized and replated into 24-well plates and cultured for 24 h before mock infection or infection with wild-type Ad5 at an MOI of 10 FFU/cell. Cells were harvested at 6 and 24 hpi, and protein lysates were analyzed by SDS-PAGE and Western blotting with antibodies to E1A, late proteins, p53, or actin. For each blot, the protein(s) of interest is highlighted with arrows. (B) As for panel A, but infections were performed at a multiplicity of infection of either 1 or 10 FFU/cell and harvested at 24 hpi. (C) MRC5 cells were transfected with control or p53 siRNA. Forty-eight hours later, cells were infected with wild-type Ad5 at an MOI of 10 FFU/cell. Cells were harvested 24 hpi, and protein lysates were analyzed by SDS-PAGE and Western blotting with antibodies to late proteins, p53, or β-actin. Protein size markers are on the left of each panel, in kDa.
Fig 6
Fig 6
L4-22K and L4-33K inhibit L4P activity. (A and B) 293 cells were transfected with either pGL-3-Basic (no promoter) or with core L4P reporter plasmid plus plasmids expressing the proteins indicated below the graph. After 24 h, cells were assayed for reporter activity (RLA), and data were expressed relative to the activity of the promoterless control. Error bars indicate the standard deviations for three biological replicates. (C) 293 cells were transfected with full-length L4P reporter plasmid plus plasmids expressing the indicated proteins for 24 h before being harvested for ChIP analysis. Samples were subjected to ChIP analysis to determine p53 binding, expressed as the percentage of L4P DNA present in the lysate (see Materials and Methods). Error bars indicate the standard deviations for three technical replicates, and data are representative of two independent repeat experiments.
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