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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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HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulateE1A promoter activation.(A) H1299 cells were transfected with luciferase reporter plasmids codingfor E1A promoter and effector plasmids encoding for VI-wt, VI-M1,VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control.Forty eight hours after transfection, samples were lysed and luciferaseactivity was measured as described before. Mean and standard deviationare from three independent experiments. (B) H1299 cells wereco-transfected with plasmids containing the Ad5 E1-region (pPG-S3) andexpression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cellextracts were prepared 48 h after transfection and proteins weresubjected to IB using Ab against RFP (pVI), pp71 or β-actin asindicated on the right. Note that several splice variants of E1A arerecognized depicted by the vertical bar. (C) Cells were transfected asin B and indicated in the legend to C. Forty eight hours aftertransfection total RNA was prepared from cell lysates and reversetranscribed using oligo-dT primers. E1A mRNA levels were determinedusing qPCR with E1A specific, exon-spanning primers. Values correspondto the mean of two experiments done in triplicates and the error barindicates the STD.
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ppat-1002549-g008: HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulateE1A promoter activation.(A) H1299 cells were transfected with luciferase reporter plasmids codingfor E1A promoter and effector plasmids encoding for VI-wt, VI-M1,VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control.Forty eight hours after transfection, samples were lysed and luciferaseactivity was measured as described before. Mean and standard deviationare from three independent experiments. (B) H1299 cells wereco-transfected with plasmids containing the Ad5 E1-region (pPG-S3) andexpression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cellextracts were prepared 48 h after transfection and proteins weresubjected to IB using Ab against RFP (pVI), pp71 or β-actin asindicated on the right. Note that several splice variants of E1A arerecognized depicted by the vertical bar. (C) Cells were transfected asin B and indicated in the legend to C. Forty eight hours aftertransfection total RNA was prepared from cell lysates and reversetranscribed using oligo-dT primers. E1A mRNA levels were determinedusing qPCR with E1A specific, exon-spanning primers. Values correspondto the mean of two experiments done in triplicates and the error barindicates the STD.

Mentions: Our data showed that protein VI activates the Ad E1 promoters by reversing Daxxrepression, presumably until newly synthesized E1A can secure the Ad geneexpression program. In this case, virion proteins derived from other DNA virusesknown to abrogate Daxx repression should be able to substitute this function. Totest this possibility, we tested whether the expression from the E1A promotercan be activated by the HCMV pp71 tegument protein or by the HPV L2 minor capsidprotein, which both target Daxx [26], [44]. Similar to protein VI-wt, pp71 and L2 were able tostimulate the Ad E1A promoter (Figure 8A). Furthermore, we observed that like protein VI-wt, pp71and L2 could also drive efficient E1A and E1B expression from a subviralconstruct, preserving the virus context encoding the E1A and E1B transcriptionunits (Figure 8B, lane 3, 6and 7). These results show that non-adenoviral virion proteins are also capableof inducing immediate early adenoviral gene expression in the absence of anyfurther Ad protein. This induction of gene expression was through mediatingtranscriptional activation, as shown by elevated E1A and E1B mRNA levels (Figure 8C). Similarly, thisresult confirmed that elevated E1A mRNA and protein expression levels driven byprotein VI require the PPxY motif, thus directly linking entry and early viralgene expression (Figure 8B,lanes 1–4). To extend the analysis for other regions of protein VI, weused the expression construct encoding protein VI-delta54, lacking theamphipathic helix, which is required to target protein VI to PML-NBs (FigureS3d). The results showed that like protein VI-M1, the constructexpressing VI-delta54 only marginally stimulated the E1A promoter (compare wt-,M1 and delta54 in Figure 8A andC). In contrast, the expression of protein VI-delta54 resulted insomewhat elevated protein expression levels compared to VI-M1 suggesting that itmight promote E1A expression on a post-transcriptional level. This could resultfrom the diffuse localization of VI-delta54 in the nucleoplasm of transfectedcells (compare with Figure S3). In summary, this analysis showedthat efficient transcriptional activation of the E1A promoter requires theamphipathic helix in addition to the PPxY motif.


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)

HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulateE1A promoter activation.(A) H1299 cells were transfected with luciferase reporter plasmids codingfor E1A promoter and effector plasmids encoding for VI-wt, VI-M1,VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control.Forty eight hours after transfection, samples were lysed and luciferaseactivity was measured as described before. Mean and standard deviationare from three independent experiments. (B) H1299 cells wereco-transfected with plasmids containing the Ad5 E1-region (pPG-S3) andexpression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cellextracts were prepared 48 h after transfection and proteins weresubjected to IB using Ab against RFP (pVI), pp71 or β-actin asindicated on the right. Note that several splice variants of E1A arerecognized depicted by the vertical bar. (C) Cells were transfected asin B and indicated in the legend to C. Forty eight hours aftertransfection total RNA was prepared from cell lysates and reversetranscribed using oligo-dT primers. E1A mRNA levels were determinedusing qPCR with E1A specific, exon-spanning primers. Values correspondto the mean of two experiments done in triplicates and the error barindicates the STD.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002549-g008: HCMV tegument protein pp71 and HPV minor capsid protein L2 stimulateE1A promoter activation.(A) H1299 cells were transfected with luciferase reporter plasmids codingfor E1A promoter and effector plasmids encoding for VI-wt, VI-M1,VI-delta54, HCMV pp71, HPV L2 or an empty vector as negative control.Forty eight hours after transfection, samples were lysed and luciferaseactivity was measured as described before. Mean and standard deviationare from three independent experiments. (B) H1299 cells wereco-transfected with plasmids containing the Ad5 E1-region (pPG-S3) andexpression vector for VI-wt, VI-M1, VI-delta54, pp71 or L2. Total-cellextracts were prepared 48 h after transfection and proteins weresubjected to IB using Ab against RFP (pVI), pp71 or β-actin asindicated on the right. Note that several splice variants of E1A arerecognized depicted by the vertical bar. (C) Cells were transfected asin B and indicated in the legend to C. Forty eight hours aftertransfection total RNA was prepared from cell lysates and reversetranscribed using oligo-dT primers. E1A mRNA levels were determinedusing qPCR with E1A specific, exon-spanning primers. Values correspondto the mean of two experiments done in triplicates and the error barindicates the STD.
Mentions: Our data showed that protein VI activates the Ad E1 promoters by reversing Daxxrepression, presumably until newly synthesized E1A can secure the Ad geneexpression program. In this case, virion proteins derived from other DNA virusesknown to abrogate Daxx repression should be able to substitute this function. Totest this possibility, we tested whether the expression from the E1A promotercan be activated by the HCMV pp71 tegument protein or by the HPV L2 minor capsidprotein, which both target Daxx [26], [44]. Similar to protein VI-wt, pp71 and L2 were able tostimulate the Ad E1A promoter (Figure 8A). Furthermore, we observed that like protein VI-wt, pp71and L2 could also drive efficient E1A and E1B expression from a subviralconstruct, preserving the virus context encoding the E1A and E1B transcriptionunits (Figure 8B, lane 3, 6and 7). These results show that non-adenoviral virion proteins are also capableof inducing immediate early adenoviral gene expression in the absence of anyfurther Ad protein. This induction of gene expression was through mediatingtranscriptional activation, as shown by elevated E1A and E1B mRNA levels (Figure 8C). Similarly, thisresult confirmed that elevated E1A mRNA and protein expression levels driven byprotein VI require the PPxY motif, thus directly linking entry and early viralgene expression (Figure 8B,lanes 1–4). To extend the analysis for other regions of protein VI, weused the expression construct encoding protein VI-delta54, lacking theamphipathic helix, which is required to target protein VI to PML-NBs (FigureS3d). The results showed that like protein VI-M1, the constructexpressing VI-delta54 only marginally stimulated the E1A promoter (compare wt-,M1 and delta54 in Figure 8A andC). In contrast, the expression of protein VI-delta54 resulted insomewhat elevated protein expression levels compared to VI-M1 suggesting that itmight promote E1A expression on a post-transcriptional level. This could resultfrom the diffuse localization of VI-delta54 in the nucleoplasm of transfectedcells (compare with Figure S3). In summary, this analysis showedthat efficient transcriptional activation of the E1A promoter requires theamphipathic helix in addition to the PPxY motif.

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