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Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo.

Mohr H, Mohr CA, Schneider MR, Scrivano L, Adler B, Kraner-Schreiber S, Schnieke A, Dahlhoff M, Wolf E, Koszinowski UH, Ruzsics Z - PLoS Pathog. (2012)

Bottom Line: This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV.The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth.Several applications are discussed.

View Article: PubMed Central - PubMed

Affiliation: Max von Pettenkofer-Institute, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.

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Related in: MedlinePlus

Infection with MCMV reactivates silenced replicon vector encoded reporter gene expression.(A) In two stable NIH3T3 cell pools transfected with pEpibo-luc-ori (luc-ori t1, luc-ori t2) expression of FL was measured in absence of infection (plain bars, - MCMV) at the indicated weeks after transfection. Reporter gene expression is lost in uninfected cells over time. At the same time points the pools were infected with MCMV at an MOI of 0.5 (hatched bars, +MCMV). 24 h p.i. of both luc-ori t1 and t2 with high expression of FL was induced. NIH3T3 fibroblasts served as a control to determine background signal (BG) of the luciferin substrate. (B) Vector pEpibo-luc-ori was inactivated by histone deacetylation. Four cell clones (cl. 1–4) derived from subcloning of luc-ori t1 were subjected to treatment with 25 µM 5′ aza-cytidine (5′Aza, gray bars), an inhibitor of CpG-methylation, 330 nM Trichostatin A (TSA, black bars) for 36 h or left untreated (mock, open bars). FL expression was analyzed in comparison to parental NIH3T3 cells. FL expression was significantly enhanced by de-condensing histone packaging through TSA treatment (*** p<0.001, ns p>0.05, Two-Way-Anova, depicted is mean+SD). RLU (relative light units), p.i. post infection, weeks = weeks post transfection of pEpibo-luc-ori.
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ppat-1002728-g001: Infection with MCMV reactivates silenced replicon vector encoded reporter gene expression.(A) In two stable NIH3T3 cell pools transfected with pEpibo-luc-ori (luc-ori t1, luc-ori t2) expression of FL was measured in absence of infection (plain bars, - MCMV) at the indicated weeks after transfection. Reporter gene expression is lost in uninfected cells over time. At the same time points the pools were infected with MCMV at an MOI of 0.5 (hatched bars, +MCMV). 24 h p.i. of both luc-ori t1 and t2 with high expression of FL was induced. NIH3T3 fibroblasts served as a control to determine background signal (BG) of the luciferin substrate. (B) Vector pEpibo-luc-ori was inactivated by histone deacetylation. Four cell clones (cl. 1–4) derived from subcloning of luc-ori t1 were subjected to treatment with 25 µM 5′ aza-cytidine (5′Aza, gray bars), an inhibitor of CpG-methylation, 330 nM Trichostatin A (TSA, black bars) for 36 h or left untreated (mock, open bars). FL expression was analyzed in comparison to parental NIH3T3 cells. FL expression was significantly enhanced by de-condensing histone packaging through TSA treatment (*** p<0.001, ns p>0.05, Two-Way-Anova, depicted is mean+SD). RLU (relative light units), p.i. post infection, weeks = weeks post transfection of pEpibo-luc-ori.

Mentions: To characterize the gene expression driven by pEpibo-luc-ori, two independent pools of NIH3T3 transfectants were generated. In the absence of infection, FL expression decreased to the limit of detection after 16 weeks in both pools (luc-ori-t1 and -t2). However, infection with MCMV restored FL expression by 3 to 4 orders of magnitude in both transgenic cell pools, irrespectively of the treatment time (Fig. 1A). Thus, loss of FL expression due to silencing was recovered upon virus infection.


Cytomegalovirus replicon-based regulation of gene expression in vitro and in vivo.

Mohr H, Mohr CA, Schneider MR, Scrivano L, Adler B, Kraner-Schreiber S, Schnieke A, Dahlhoff M, Wolf E, Koszinowski UH, Ruzsics Z - PLoS Pathog. (2012)

Infection with MCMV reactivates silenced replicon vector encoded reporter gene expression.(A) In two stable NIH3T3 cell pools transfected with pEpibo-luc-ori (luc-ori t1, luc-ori t2) expression of FL was measured in absence of infection (plain bars, - MCMV) at the indicated weeks after transfection. Reporter gene expression is lost in uninfected cells over time. At the same time points the pools were infected with MCMV at an MOI of 0.5 (hatched bars, +MCMV). 24 h p.i. of both luc-ori t1 and t2 with high expression of FL was induced. NIH3T3 fibroblasts served as a control to determine background signal (BG) of the luciferin substrate. (B) Vector pEpibo-luc-ori was inactivated by histone deacetylation. Four cell clones (cl. 1–4) derived from subcloning of luc-ori t1 were subjected to treatment with 25 µM 5′ aza-cytidine (5′Aza, gray bars), an inhibitor of CpG-methylation, 330 nM Trichostatin A (TSA, black bars) for 36 h or left untreated (mock, open bars). FL expression was analyzed in comparison to parental NIH3T3 cells. FL expression was significantly enhanced by de-condensing histone packaging through TSA treatment (*** p<0.001, ns p>0.05, Two-Way-Anova, depicted is mean+SD). RLU (relative light units), p.i. post infection, weeks = weeks post transfection of pEpibo-luc-ori.
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Related In: Results  -  Collection

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ppat-1002728-g001: Infection with MCMV reactivates silenced replicon vector encoded reporter gene expression.(A) In two stable NIH3T3 cell pools transfected with pEpibo-luc-ori (luc-ori t1, luc-ori t2) expression of FL was measured in absence of infection (plain bars, - MCMV) at the indicated weeks after transfection. Reporter gene expression is lost in uninfected cells over time. At the same time points the pools were infected with MCMV at an MOI of 0.5 (hatched bars, +MCMV). 24 h p.i. of both luc-ori t1 and t2 with high expression of FL was induced. NIH3T3 fibroblasts served as a control to determine background signal (BG) of the luciferin substrate. (B) Vector pEpibo-luc-ori was inactivated by histone deacetylation. Four cell clones (cl. 1–4) derived from subcloning of luc-ori t1 were subjected to treatment with 25 µM 5′ aza-cytidine (5′Aza, gray bars), an inhibitor of CpG-methylation, 330 nM Trichostatin A (TSA, black bars) for 36 h or left untreated (mock, open bars). FL expression was analyzed in comparison to parental NIH3T3 cells. FL expression was significantly enhanced by de-condensing histone packaging through TSA treatment (*** p<0.001, ns p>0.05, Two-Way-Anova, depicted is mean+SD). RLU (relative light units), p.i. post infection, weeks = weeks post transfection of pEpibo-luc-ori.
Mentions: To characterize the gene expression driven by pEpibo-luc-ori, two independent pools of NIH3T3 transfectants were generated. In the absence of infection, FL expression decreased to the limit of detection after 16 weeks in both pools (luc-ori-t1 and -t2). However, infection with MCMV restored FL expression by 3 to 4 orders of magnitude in both transgenic cell pools, irrespectively of the treatment time (Fig. 1A). Thus, loss of FL expression due to silencing was recovered upon virus infection.

Bottom Line: This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV.The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth.Several applications are discussed.

View Article: PubMed Central - PubMed

Affiliation: Max von Pettenkofer-Institute, Ludwig-Maximilians-Universität München, Munich, Germany.

ABSTRACT
There is increasing evidence for a connection between DNA replication and the expression of adjacent genes. Therefore, this study addressed the question of whether a herpesvirus origin of replication can be used to activate or increase the expression of adjacent genes. Cell lines carrying an episomal vector, in which reporter genes are linked to the murine cytomegalovirus (MCMV) origin of lytic replication (oriLyt), were constructed. Reporter gene expression was silenced by a histone-deacetylase-dependent mechanism, but was resolved upon lytic infection with MCMV. Replication of the episome was observed subsequent to infection, leading to the induction of gene expression by more than 1000-fold. oriLyt-based regulation thus provided a unique opportunity for virus-induced conditional gene expression without the need for an additional induction mechanism. This principle was exploited to show effective late trans-complementation of the toxic viral protein M50 and the glycoprotein gO of MCMV. Moreover, the application of this principle for intracellular immunization against herpesvirus infection was demonstrated. The results of the present study show that viral infection specifically activated the expression of a dominant-negative transgene, which inhibited viral growth. This conditional system was operative in explant cultures of transgenic mice, but not in vivo. Several applications are discussed.

Show MeSH
Related in: MedlinePlus