Limits...
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.

Show MeSH

Related in: MedlinePlus

Viruses with mutated PPxY motif in protein VI (M1) have altered geneexpression.(A) U2OS cells were infected with replication competent HH-Ad5-VI-wt orHH-Ad5-VI-M1 with a multiplicity of infection (MOI) of 50 FFU/cell (seeFigure S1). Viral particles were harvested at 24, 48 and 72h p.i. and virus yield was determined by quantitative E2A stain. Theresults represent the average from three independent experiments. (B)U2OS cells were infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at a MOI of50 FFU/cell and whole-cell extracts were prepared after indicatedtime-points and subjected to immunoblotting (IB). Corresponding proteinsare indicated to the right. MOI dependent replication differences areshown in Figure S2. (C) Cells were infected as in A and newlysynthesized RNAs were labelled for the time p.i. as indicated on thex-axis and described in the materials andmethods section. Extracted RNAs were reverse transcribed andquantified using qPCR using exon-spanning E1A specific primers andnormalized against GAPDH mRNA levels. E1A mRNA levels in wt-infectedcells were arbitrarily set to 100%. Data are derived from threeindependent experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303589&req=5

ppat-1002549-g001: Viruses with mutated PPxY motif in protein VI (M1) have altered geneexpression.(A) U2OS cells were infected with replication competent HH-Ad5-VI-wt orHH-Ad5-VI-M1 with a multiplicity of infection (MOI) of 50 FFU/cell (seeFigure S1). Viral particles were harvested at 24, 48 and 72h p.i. and virus yield was determined by quantitative E2A stain. Theresults represent the average from three independent experiments. (B)U2OS cells were infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at a MOI of50 FFU/cell and whole-cell extracts were prepared after indicatedtime-points and subjected to immunoblotting (IB). Corresponding proteinsare indicated to the right. MOI dependent replication differences areshown in Figure S2. (C) Cells were infected as in A and newlysynthesized RNAs were labelled for the time p.i. as indicated on thex-axis and described in the materials andmethods section. Extracted RNAs were reverse transcribed andquantified using qPCR using exon-spanning E1A specific primers andnormalized against GAPDH mRNA levels. E1A mRNA levels in wt-infectedcells were arbitrarily set to 100%. Data are derived from threeindependent experiments.

Mentions: To investigate the role of protein VI during post-endosomolytic steps requiredfor the onset of viral replication, we constructed replication competent Adscontaining the E1 region with either wildtype (wt) protein VI (HH-Ad5-VI-wt,depicted in the Figure S1) or mutant “M1” protein VI in which the PPSYmotif was mutated to PGAA that abolished Nedd4 interaction [HH-Ad5-M1;Fig.S1; [36]]. Following infection of U2OS cells, we observedthat M1 virus replication was attenuated compared to wt (Figure 1A and S1B). Thisis in agreement with our previous observations showing reduced infectivity of anE1-deleted M1 Ad vector compared to the corresponding E1-deleted wt Ad vector[36]. Todistinguish between capsid transport and possible more downstream effects, weinfected cells with different amounts of replication competent wt and M1viruses. Then, we determined the genome copy numbers in nuclear and cytoplasmicfractions by qPCR and the efficiency of the initiation of virus replication byquantification of E2A stained replication centers (detailed in Figure S2).Compared to wt, fewer M1 virus genomes accumulated in the nucleus associatedfraction, independent of the amount of input virus. In contrast, initiation ofvirus replication for M1 genomes was reduced for low, but not at high physicalparticle per cell ratios (Figure S2) suggesting defects downstream ofvirus nuclear transport.


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)

Viruses with mutated PPxY motif in protein VI (M1) have altered geneexpression.(A) U2OS cells were infected with replication competent HH-Ad5-VI-wt orHH-Ad5-VI-M1 with a multiplicity of infection (MOI) of 50 FFU/cell (seeFigure S1). Viral particles were harvested at 24, 48 and 72h p.i. and virus yield was determined by quantitative E2A stain. Theresults represent the average from three independent experiments. (B)U2OS cells were infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at a MOI of50 FFU/cell and whole-cell extracts were prepared after indicatedtime-points and subjected to immunoblotting (IB). Corresponding proteinsare indicated to the right. MOI dependent replication differences areshown in Figure S2. (C) Cells were infected as in A and newlysynthesized RNAs were labelled for the time p.i. as indicated on thex-axis and described in the materials andmethods section. Extracted RNAs were reverse transcribed andquantified using qPCR using exon-spanning E1A specific primers andnormalized against GAPDH mRNA levels. E1A mRNA levels in wt-infectedcells were arbitrarily set to 100%. Data are derived from threeindependent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002549-g001: Viruses with mutated PPxY motif in protein VI (M1) have altered geneexpression.(A) U2OS cells were infected with replication competent HH-Ad5-VI-wt orHH-Ad5-VI-M1 with a multiplicity of infection (MOI) of 50 FFU/cell (seeFigure S1). Viral particles were harvested at 24, 48 and 72h p.i. and virus yield was determined by quantitative E2A stain. Theresults represent the average from three independent experiments. (B)U2OS cells were infected with HH-Ad5-VI-wt or HH-Ad5-VI-M1 at a MOI of50 FFU/cell and whole-cell extracts were prepared after indicatedtime-points and subjected to immunoblotting (IB). Corresponding proteinsare indicated to the right. MOI dependent replication differences areshown in Figure S2. (C) Cells were infected as in A and newlysynthesized RNAs were labelled for the time p.i. as indicated on thex-axis and described in the materials andmethods section. Extracted RNAs were reverse transcribed andquantified using qPCR using exon-spanning E1A specific primers andnormalized against GAPDH mRNA levels. E1A mRNA levels in wt-infectedcells were arbitrarily set to 100%. Data are derived from threeindependent experiments.
Mentions: To investigate the role of protein VI during post-endosomolytic steps requiredfor the onset of viral replication, we constructed replication competent Adscontaining the E1 region with either wildtype (wt) protein VI (HH-Ad5-VI-wt,depicted in the Figure S1) or mutant “M1” protein VI in which the PPSYmotif was mutated to PGAA that abolished Nedd4 interaction [HH-Ad5-M1;Fig.S1; [36]]. Following infection of U2OS cells, we observedthat M1 virus replication was attenuated compared to wt (Figure 1A and S1B). Thisis in agreement with our previous observations showing reduced infectivity of anE1-deleted M1 Ad vector compared to the corresponding E1-deleted wt Ad vector[36]. Todistinguish between capsid transport and possible more downstream effects, weinfected cells with different amounts of replication competent wt and M1viruses. Then, we determined the genome copy numbers in nuclear and cytoplasmicfractions by qPCR and the efficiency of the initiation of virus replication byquantification of E2A stained replication centers (detailed in Figure S2).Compared to wt, fewer M1 virus genomes accumulated in the nucleus associatedfraction, independent of the amount of input virus. In contrast, initiation ofvirus replication for M1 genomes was reduced for low, but not at high physicalparticle per cell ratios (Figure S2) suggesting defects downstream ofvirus nuclear transport.

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