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Transcriptional activation of the adenoviral genome is mediated by capsid protein VI.

Schreiner S, Martinez R, Groitl P, Rayne F, Vaillant R, Wimmer P, Bossis G, Sternsdorf T, Marcinowski L, Ruzsics Z, Dobner T, Wodrich H - PLoS Pathog. (2012)

Bottom Line: Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI.This requires a conserved PPxY motif in protein VI.Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.

ABSTRACT
Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins.

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Protein VI mediates Ad transcriptional activation.(A) H1299 cells were transfected with luciferase reporter plasmids underE1A promoter- (left panel), E1B promoter- (right panel) or promoterlesscontrol and effector plasmids expressing VI-wt or VI-M1. Forty eighthours after transfection, samples were lysed, absolute luciferaseactivity was measured and activity of the E1 promoter alone wasnormalized to 100%. Mean and STD are from three independentexperiments. Effects on additional viral promoters are shown in FigureS4. (B) H1299 cells were infected with HH-Ad5-VI-wt orHH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Forty eight hours p.i., cells werefixed with formaldehyde and ChIP analysis was performed as described inmaterials and methods. Theaverage Ct-value was determined from triplicate reactions andnormalized with standard curves for each primer pair. The y-axisindicates the percentage of immunoprecipitated signal from the input( = 100%). (C) HAD cells were transfectedwith E1B promoter constructs and effector plasmids encoding for Daxx,VI-wt, VI-M1. Forty eight hours after transfection, samples were lysedand analyzed as in (A). Mean and STD are from three independentexperiments.
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ppat-1002549-g004: Protein VI mediates Ad transcriptional activation.(A) H1299 cells were transfected with luciferase reporter plasmids underE1A promoter- (left panel), E1B promoter- (right panel) or promoterlesscontrol and effector plasmids expressing VI-wt or VI-M1. Forty eighthours after transfection, samples were lysed, absolute luciferaseactivity was measured and activity of the E1 promoter alone wasnormalized to 100%. Mean and STD are from three independentexperiments. Effects on additional viral promoters are shown in FigureS4. (B) H1299 cells were infected with HH-Ad5-VI-wt orHH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Forty eight hours p.i., cells werefixed with formaldehyde and ChIP analysis was performed as described inmaterials and methods. Theaverage Ct-value was determined from triplicate reactions andnormalized with standard curves for each primer pair. The y-axisindicates the percentage of immunoprecipitated signal from the input( = 100%). (C) HAD cells were transfectedwith E1B promoter constructs and effector plasmids encoding for Daxx,VI-wt, VI-M1. Forty eight hours after transfection, samples were lysedand analyzed as in (A). Mean and STD are from three independentexperiments.

Mentions: Next, we asked whether the Ad immediate early E1A and early E1B promoters aretargeted by Daxx mediated repression and if this is the case whether it can bereversed by protein VI. To this end, we constructed luciferase expressionvectors controlled by the Ad E1A and E1B promoters and measured luciferaseexpression in protein VI-wt or protein VI-M1 transfected H1299 cells (Figure 4A). Unlike VI-M1,VI-wt was able to stimulate expression from the E1A promoter ∼2.5-fold and∼1.5-fold from the E1B promoter (Figure 4A). To show direct association ofprotein VI with E1 promoters, we performed chromatin immunoprecipitation assays(ChIP) at 48 h p.i from M1- or wt virus infected cells, using protein VIspecific serum and Ad promoter-specific primers (Figure 4B). The results show that the VI-wtprotein was much more strongly associated with the E1A and E1B promoter ininfected cells than the VI-M1 protein, which is also reflected in their relativeactivation ability (Figure4B, compare with 4A). To analyze whether protein VI associatedactivation of Ad early promoters is involved in Daxx de-repression, wecotransfected the E1B promoter driven luciferase expression vector in absence orpresence of Daxx with protein VI-wt or VI-M1 expression vectors. Protein VI-wt,but not VI-M1, alleviated Daxx repression implying a role for the PPxY motif(Figure 4C). Althoughthere was less binding to protein VI compared to the E1A promoter, we observed astrong effect on the activation of luciferase expression in that experiment. Wealso tested if protein VI (wt or M1) stimulates other Ad promoters usingluciferase expression vectors for all viral promoters. The data showed thatprotein VI-wt was able to stimulate most of the Ad promoters in absence of otherviral factors to various degrees (Figure S4). The strongest induction wasobserved for the immediate early E1A and E2A early promoter, which is inagreement with the weak E2A expression observed in HepaRG cells in M1-virusinfected cells and the restoration of E2A expression following Daxx depletion(see Figure 3E). Incontrast, E3 and E4 promoter activation was weak with no clear differencebetween wt and M1. In the context of an ongoing virus infection, thetranscriptional activation of both promoter groups (E1/E2 vs. E3/E4) was shownto be regulated by E1A but via different mechanisms [40], [41]. Thus, ourdata showed that protein VI might also play a minor role in the transcriptionalactivation of the E1/E2 promoter group.


Transcriptional activation of the adenoviral genome is mediated by capsid protein VI.

Schreiner S, Martinez R, Groitl P, Rayne F, Vaillant R, Wimmer P, Bossis G, Sternsdorf T, Marcinowski L, Ruzsics Z, Dobner T, Wodrich H - PLoS Pathog. (2012)

Protein VI mediates Ad transcriptional activation.(A) H1299 cells were transfected with luciferase reporter plasmids underE1A promoter- (left panel), E1B promoter- (right panel) or promoterlesscontrol and effector plasmids expressing VI-wt or VI-M1. Forty eighthours after transfection, samples were lysed, absolute luciferaseactivity was measured and activity of the E1 promoter alone wasnormalized to 100%. Mean and STD are from three independentexperiments. Effects on additional viral promoters are shown in FigureS4. (B) H1299 cells were infected with HH-Ad5-VI-wt orHH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Forty eight hours p.i., cells werefixed with formaldehyde and ChIP analysis was performed as described inmaterials and methods. Theaverage Ct-value was determined from triplicate reactions andnormalized with standard curves for each primer pair. The y-axisindicates the percentage of immunoprecipitated signal from the input( = 100%). (C) HAD cells were transfectedwith E1B promoter constructs and effector plasmids encoding for Daxx,VI-wt, VI-M1. Forty eight hours after transfection, samples were lysedand analyzed as in (A). Mean and STD are from three independentexperiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303589&req=5

ppat-1002549-g004: Protein VI mediates Ad transcriptional activation.(A) H1299 cells were transfected with luciferase reporter plasmids underE1A promoter- (left panel), E1B promoter- (right panel) or promoterlesscontrol and effector plasmids expressing VI-wt or VI-M1. Forty eighthours after transfection, samples were lysed, absolute luciferaseactivity was measured and activity of the E1 promoter alone wasnormalized to 100%. Mean and STD are from three independentexperiments. Effects on additional viral promoters are shown in FigureS4. (B) H1299 cells were infected with HH-Ad5-VI-wt orHH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Forty eight hours p.i., cells werefixed with formaldehyde and ChIP analysis was performed as described inmaterials and methods. Theaverage Ct-value was determined from triplicate reactions andnormalized with standard curves for each primer pair. The y-axisindicates the percentage of immunoprecipitated signal from the input( = 100%). (C) HAD cells were transfectedwith E1B promoter constructs and effector plasmids encoding for Daxx,VI-wt, VI-M1. Forty eight hours after transfection, samples were lysedand analyzed as in (A). Mean and STD are from three independentexperiments.
Mentions: Next, we asked whether the Ad immediate early E1A and early E1B promoters aretargeted by Daxx mediated repression and if this is the case whether it can bereversed by protein VI. To this end, we constructed luciferase expressionvectors controlled by the Ad E1A and E1B promoters and measured luciferaseexpression in protein VI-wt or protein VI-M1 transfected H1299 cells (Figure 4A). Unlike VI-M1,VI-wt was able to stimulate expression from the E1A promoter ∼2.5-fold and∼1.5-fold from the E1B promoter (Figure 4A). To show direct association ofprotein VI with E1 promoters, we performed chromatin immunoprecipitation assays(ChIP) at 48 h p.i from M1- or wt virus infected cells, using protein VIspecific serum and Ad promoter-specific primers (Figure 4B). The results show that the VI-wtprotein was much more strongly associated with the E1A and E1B promoter ininfected cells than the VI-M1 protein, which is also reflected in their relativeactivation ability (Figure4B, compare with 4A). To analyze whether protein VI associatedactivation of Ad early promoters is involved in Daxx de-repression, wecotransfected the E1B promoter driven luciferase expression vector in absence orpresence of Daxx with protein VI-wt or VI-M1 expression vectors. Protein VI-wt,but not VI-M1, alleviated Daxx repression implying a role for the PPxY motif(Figure 4C). Althoughthere was less binding to protein VI compared to the E1A promoter, we observed astrong effect on the activation of luciferase expression in that experiment. Wealso tested if protein VI (wt or M1) stimulates other Ad promoters usingluciferase expression vectors for all viral promoters. The data showed thatprotein VI-wt was able to stimulate most of the Ad promoters in absence of otherviral factors to various degrees (Figure S4). The strongest induction wasobserved for the immediate early E1A and E2A early promoter, which is inagreement with the weak E2A expression observed in HepaRG cells in M1-virusinfected cells and the restoration of E2A expression following Daxx depletion(see Figure 3E). Incontrast, E3 and E4 promoter activation was weak with no clear differencebetween wt and M1. In the context of an ongoing virus infection, thetranscriptional activation of both promoter groups (E1/E2 vs. E3/E4) was shownto be regulated by E1A but via different mechanisms [40], [41]. Thus, ourdata showed that protein VI might also play a minor role in the transcriptionalactivation of the E1/E2 promoter group.

Bottom Line: Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI.This requires a conserved PPxY motif in protein VI.Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany.

ABSTRACT
Gene expression of DNA viruses requires nuclear import of the viral genome. Human Adenoviruses (Ads), like most DNA viruses, encode factors within early transcription units promoting their own gene expression and counteracting cellular antiviral defense mechanisms. The cellular transcriptional repressor Daxx prevents viral gene expression through the assembly of repressive chromatin remodeling complexes targeting incoming viral genomes. However, it has remained unclear how initial transcriptional activation of the adenoviral genome is achieved. Here we show that Daxx mediated repression of the immediate early Ad E1A promoter is efficiently counteracted by the capsid protein VI. This requires a conserved PPxY motif in protein VI. Capsid proteins from other DNA viruses were also shown to activate the Ad E1A promoter independent of Ad gene expression and support virus replication. Our results show how Ad entry is connected to transcriptional activation of their genome in the nucleus. Our data further suggest a common principle for genome activation of DNA viruses by counteracting Daxx related repressive mechanisms through virion proteins.

Show MeSH
Related in: MedlinePlus