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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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pEpibo-luc-ori is amplified upon MCMV infection.luc-ori cl.1 cells were infected with MCMV (white hatched bars), MHV68 (black hatched bars) at an MOI of 1 or left untreated (white bar, mock), or treated with 330 nM TSA. In addition, the DNA replication inhibitors PAA (300 µg/ml) and PF (100 µg/ml) were added to infected cells. (A) Bioluminescence assay was performed to determine the FL induction and (B) quantitative realtime PCR was performed 36 h p.i. to determine copy numbers of pEpibo-luc-ori vectors by a PCR specific for the bsr coding sequence compared to the cellular gene lamin B receptor (lbr).
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ppat-1002728-g003: pEpibo-luc-ori is amplified upon MCMV infection.luc-ori cl.1 cells were infected with MCMV (white hatched bars), MHV68 (black hatched bars) at an MOI of 1 or left untreated (white bar, mock), or treated with 330 nM TSA. In addition, the DNA replication inhibitors PAA (300 µg/ml) and PF (100 µg/ml) were added to infected cells. (A) Bioluminescence assay was performed to determine the FL induction and (B) quantitative realtime PCR was performed 36 h p.i. to determine copy numbers of pEpibo-luc-ori vectors by a PCR specific for the bsr coding sequence compared to the cellular gene lamin B receptor (lbr).

Mentions: Because induction of reporter gene expression from the vector was dependent on the oriLyt sequence (Fig. 2B), quantitative PCR (qPCR) experiments were performed to determine whether the episomal vector was amplified during infection. We measured the relative pEpibo-luc-ori copy numbers in infected and uninfected cells normalized to the endogenous murine lbr gene. Induction of FL expression (Fig. 3A) correlated with a significant increase in the pEpibo-luc-ori copy number (about 50-fold; Fig. 3B). Notably, in the cell line, luc-ori cl.4, in which MCMV infection did not de-silence FL expression, there was no increase in the copy number of the replicon vector sequence (Supp Fig. S5). Phosphonoformic acid (PF), like PAA, is a specific inhibitor of herpesvirus DNA polymerases. As expected, no amplification of the vector was detected after PAA or PF treatment of infected luc-ori cl.1 cells (Fig. 3B), whereas infection of luc-ori cl.1 cells in the absence of inhibitors resulted in a ∼2500-fold increase in FL induction and a ∼50-fold increase in replicon vector amplification. By contrast, liberation of the vector from silencing by treatment with 300 nM TSA, without additional vector replication, resulted in only a moderate ∼25–30 fold induction of FL in luc-ori cl.1 cells (Fig. 3A, B).


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)

pEpibo-luc-ori is amplified upon MCMV infection.luc-ori cl.1 cells were infected with MCMV (white hatched bars), MHV68 (black hatched bars) at an MOI of 1 or left untreated (white bar, mock), or treated with 330 nM TSA. In addition, the DNA replication inhibitors PAA (300 µg/ml) and PF (100 µg/ml) were added to infected cells. (A) Bioluminescence assay was performed to determine the FL induction and (B) quantitative realtime PCR was performed 36 h p.i. to determine copy numbers of pEpibo-luc-ori vectors by a PCR specific for the bsr coding sequence compared to the cellular gene lamin B receptor (lbr).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369935&req=5

ppat-1002728-g003: pEpibo-luc-ori is amplified upon MCMV infection.luc-ori cl.1 cells were infected with MCMV (white hatched bars), MHV68 (black hatched bars) at an MOI of 1 or left untreated (white bar, mock), or treated with 330 nM TSA. In addition, the DNA replication inhibitors PAA (300 µg/ml) and PF (100 µg/ml) were added to infected cells. (A) Bioluminescence assay was performed to determine the FL induction and (B) quantitative realtime PCR was performed 36 h p.i. to determine copy numbers of pEpibo-luc-ori vectors by a PCR specific for the bsr coding sequence compared to the cellular gene lamin B receptor (lbr).
Mentions: Because induction of reporter gene expression from the vector was dependent on the oriLyt sequence (Fig. 2B), quantitative PCR (qPCR) experiments were performed to determine whether the episomal vector was amplified during infection. We measured the relative pEpibo-luc-ori copy numbers in infected and uninfected cells normalized to the endogenous murine lbr gene. Induction of FL expression (Fig. 3A) correlated with a significant increase in the pEpibo-luc-ori copy number (about 50-fold; Fig. 3B). Notably, in the cell line, luc-ori cl.4, in which MCMV infection did not de-silence FL expression, there was no increase in the copy number of the replicon vector sequence (Supp Fig. S5). Phosphonoformic acid (PF), like PAA, is a specific inhibitor of herpesvirus DNA polymerases. As expected, no amplification of the vector was detected after PAA or PF treatment of infected luc-ori cl.1 cells (Fig. 3B), whereas infection of luc-ori cl.1 cells in the absence of inhibitors resulted in a ∼2500-fold increase in FL induction and a ∼50-fold increase in replicon vector amplification. By contrast, liberation of the vector from silencing by treatment with 300 nM TSA, without additional vector replication, resulted in only a moderate ∼25–30 fold induction of FL in luc-ori cl.1 cells (Fig. 3A, B).

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