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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 cansubstitute transcriptional activation of the Ad genome.(A) H1299 cells were transfected with control vector, VI-wt, VI-M1 orVI-delta54 expression vector and subsequently infected with HH-Ad5-VI-wtor HH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Viral particles were harvested24, 48 and 72 h p.i. and virus yield was determined using quantitativeE2A stain. (B) Experimental setup as in A including the use of the sameempty vector control except that cells were transfected with expressionvector for the HCMV tegument protein pp71 or the HPV small capsidprotein L2. Results are from threeindependent experiments. (C) U2OS cells were transfected with controlvectors or expression vectors for VI-wt or VI-M1 as indicated in thelegend together with control expression vectors forRenilla luciferase. Twenty four hours aftertransfection, cells were infected with MCMV encoding a fireflyluciferase gene controlled by the HCMV immediate early promoter. Twohours after infection cells were lysed and firefly luciferase levelswere measured and normalized for renilla luciferase expression by a dualluciferase assay. Expression levels were set to 100% for emptyvectors. Results are the mean oftwo independent experiments performed in six technical repeats. Errorbars represent the STD.
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ppat-1002549-g009: HCMV tegument protein pp71 and HPV minor capsid protein L2 cansubstitute transcriptional activation of the Ad genome.(A) H1299 cells were transfected with control vector, VI-wt, VI-M1 orVI-delta54 expression vector and subsequently infected with HH-Ad5-VI-wtor HH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Viral particles were harvested24, 48 and 72 h p.i. and virus yield was determined using quantitativeE2A stain. (B) Experimental setup as in A including the use of the sameempty vector control except that cells were transfected with expressionvector for the HCMV tegument protein pp71 or the HPV small capsidprotein L2. Results are from threeindependent experiments. (C) U2OS cells were transfected with controlvectors or expression vectors for VI-wt or VI-M1 as indicated in thelegend together with control expression vectors forRenilla luciferase. Twenty four hours aftertransfection, cells were infected with MCMV encoding a fireflyluciferase gene controlled by the HCMV immediate early promoter. Twohours after infection cells were lysed and firefly luciferase levelswere measured and normalized for renilla luciferase expression by a dualluciferase assay. Expression levels were set to 100% for emptyvectors. Results are the mean oftwo independent experiments performed in six technical repeats. Errorbars represent the STD.

Mentions: Because protein VI, pp71 and L2 can stimulate Ad E1A expression independently, wenext asked if they could compensate for the lack of functional PPxY motif in thereplication competent HH-Ad5-VI-M1 virus. We transfected cells with expressionvectors for protein VI-wt, VI-M1 and VI-delta54 (Figure 9A) and HCMV tegument protein pp71 andHPV small capsid protein L2 (Figure9B) followed by infection with HH-Ad5-VI-wt or HH-Ad5-VI-M1 virus.The analysis showed that protein VI-wt was able to fully compensate for the M1mutation in the virus and restored progeny virus production to wt levels, whileprotein VI-M1 was not able to rescue virus production and VI-delta54 resultedonly in partial rescue (Figure9A). Amazingly, HCMV pp71 and HPV L2 were also fully capable ofcomplementing the M1 mutant virus and restored progeny virus production to wtlevels (Figure 9B). Lastly,we wanted to know if the adenoviral protein VI capsid protein was also able tostimulate an immediate early promoter in the context of a non-related virusinfection. We transfected U2OS cells with protein VI-wt and VI-M1 or a controlvector and infected the transfected cells with a murine cytomegalovirus (MCMV)expressing luciferase under the control of the HCMV immediate early promoter(MCMV-Luc). Luciferase expression was measured 2 h after a synchronizedinfection to quantify the activation of the immediate early promoter. Theresults showed that only protein VI-wt was able to stimulate immediate earlypromoter in the context of MCMV infection (Figure 9C).


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 cansubstitute transcriptional activation of the Ad genome.(A) H1299 cells were transfected with control vector, VI-wt, VI-M1 orVI-delta54 expression vector and subsequently infected with HH-Ad5-VI-wtor HH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Viral particles were harvested24, 48 and 72 h p.i. and virus yield was determined using quantitativeE2A stain. (B) Experimental setup as in A including the use of the sameempty vector control except that cells were transfected with expressionvector for the HCMV tegument protein pp71 or the HPV small capsidprotein L2. Results are from threeindependent experiments. (C) U2OS cells were transfected with controlvectors or expression vectors for VI-wt or VI-M1 as indicated in thelegend together with control expression vectors forRenilla luciferase. Twenty four hours aftertransfection, cells were infected with MCMV encoding a fireflyluciferase gene controlled by the HCMV immediate early promoter. Twohours after infection cells were lysed and firefly luciferase levelswere measured and normalized for renilla luciferase expression by a dualluciferase assay. Expression levels were set to 100% for emptyvectors. Results are the mean oftwo independent experiments performed in six technical repeats. Errorbars represent the STD.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002549-g009: HCMV tegument protein pp71 and HPV minor capsid protein L2 cansubstitute transcriptional activation of the Ad genome.(A) H1299 cells were transfected with control vector, VI-wt, VI-M1 orVI-delta54 expression vector and subsequently infected with HH-Ad5-VI-wtor HH-Ad5-VI-M1 at a MOI of 50 FFU/cell. Viral particles were harvested24, 48 and 72 h p.i. and virus yield was determined using quantitativeE2A stain. (B) Experimental setup as in A including the use of the sameempty vector control except that cells were transfected with expressionvector for the HCMV tegument protein pp71 or the HPV small capsidprotein L2. Results are from threeindependent experiments. (C) U2OS cells were transfected with controlvectors or expression vectors for VI-wt or VI-M1 as indicated in thelegend together with control expression vectors forRenilla luciferase. Twenty four hours aftertransfection, cells were infected with MCMV encoding a fireflyluciferase gene controlled by the HCMV immediate early promoter. Twohours after infection cells were lysed and firefly luciferase levelswere measured and normalized for renilla luciferase expression by a dualluciferase assay. Expression levels were set to 100% for emptyvectors. Results are the mean oftwo independent experiments performed in six technical repeats. Errorbars represent the STD.
Mentions: Because protein VI, pp71 and L2 can stimulate Ad E1A expression independently, wenext asked if they could compensate for the lack of functional PPxY motif in thereplication competent HH-Ad5-VI-M1 virus. We transfected cells with expressionvectors for protein VI-wt, VI-M1 and VI-delta54 (Figure 9A) and HCMV tegument protein pp71 andHPV small capsid protein L2 (Figure9B) followed by infection with HH-Ad5-VI-wt or HH-Ad5-VI-M1 virus.The analysis showed that protein VI-wt was able to fully compensate for the M1mutation in the virus and restored progeny virus production to wt levels, whileprotein VI-M1 was not able to rescue virus production and VI-delta54 resultedonly in partial rescue (Figure9A). Amazingly, HCMV pp71 and HPV L2 were also fully capable ofcomplementing the M1 mutant virus and restored progeny virus production to wtlevels (Figure 9B). Lastly,we wanted to know if the adenoviral protein VI capsid protein was also able tostimulate an immediate early promoter in the context of a non-related virusinfection. We transfected U2OS cells with protein VI-wt and VI-M1 or a controlvector and infected the transfected cells with a murine cytomegalovirus (MCMV)expressing luciferase under the control of the HCMV immediate early promoter(MCMV-Luc). Luciferase expression was measured 2 h after a synchronizedinfection to quantify the activation of the immediate early promoter. Theresults showed that only protein VI-wt was able to stimulate immediate earlypromoter in the context of MCMV infection (Figure 9C).

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