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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

Induction of replicon vector is dependent on the MCMV DNA replication.(A) Induction of MCMV oriLyt is specific to MCMV infection. Cell line luc-ori cl.1 was infected with MCMV (beta herpesvirus; MOI = 0.5) or murine herpesvirus 68 (MHV68, gamma herpesvirus; MOI = 0.5) or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (B) NIH3T3 cells were stably transfected with pEpibo-luc (luc t1) or pEpibo-luc-ori (luc-ori t3). Depicted is the ratio of FL expression of the resulting cell pools before and after infection with MCMV at an MOI of 1. In the luc t1 cell pool, lacking the oriLyt sequence, FL expression is not enhanced by infection, in contrast to the luc-ori t3 cell pool, in which FL expression is induced about 40 fold at 36 h p.i. (C) NIH3T3 or luc-ori cell lines (luc-ori cl.1–cl.4) were infected with MCMV at an MOI of 0.5 (hatched bars) or left uninfected (plain bars). In addition, cell lines were either treated with phosponoacidic acid (PAA, 300 µg/ml, black bars) or left untreated (white bars). 36 h p.i. FL induction was measured via bioluminescence assay. While reporter expression is induced up to 1000-fold in MCMV infected cells lines cl.1–3, inhibition of viral DNA polymerase by PAA blocks the induction of FL expression. (D) Induction can be inhibited with PAA completely if it is added before replication has started and can be reduced if added after the onset of DNA replication. Cell line luc-ori cl.1 was infected with MCMV at an MOI = 0.5 or left uninfected. Arrows indicate time points when 300 µg/ml PAA was added to the cells. Cells were harvested at indicated time points and FL was measured via bioluminescence assay. Induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (p.i., post infection; BG = Background; ***: p<0.001, ns: p>0.05, Two-Way-ANOVA, depicted is mean+SD).
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ppat-1002728-g002: Induction of replicon vector is dependent on the MCMV DNA replication.(A) Induction of MCMV oriLyt is specific to MCMV infection. Cell line luc-ori cl.1 was infected with MCMV (beta herpesvirus; MOI = 0.5) or murine herpesvirus 68 (MHV68, gamma herpesvirus; MOI = 0.5) or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (B) NIH3T3 cells were stably transfected with pEpibo-luc (luc t1) or pEpibo-luc-ori (luc-ori t3). Depicted is the ratio of FL expression of the resulting cell pools before and after infection with MCMV at an MOI of 1. In the luc t1 cell pool, lacking the oriLyt sequence, FL expression is not enhanced by infection, in contrast to the luc-ori t3 cell pool, in which FL expression is induced about 40 fold at 36 h p.i. (C) NIH3T3 or luc-ori cell lines (luc-ori cl.1–cl.4) were infected with MCMV at an MOI of 0.5 (hatched bars) or left uninfected (plain bars). In addition, cell lines were either treated with phosponoacidic acid (PAA, 300 µg/ml, black bars) or left untreated (white bars). 36 h p.i. FL induction was measured via bioluminescence assay. While reporter expression is induced up to 1000-fold in MCMV infected cells lines cl.1–3, inhibition of viral DNA polymerase by PAA blocks the induction of FL expression. (D) Induction can be inhibited with PAA completely if it is added before replication has started and can be reduced if added after the onset of DNA replication. Cell line luc-ori cl.1 was infected with MCMV at an MOI = 0.5 or left uninfected. Arrows indicate time points when 300 µg/ml PAA was added to the cells. Cells were harvested at indicated time points and FL was measured via bioluminescence assay. Induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (p.i., post infection; BG = Background; ***: p<0.001, ns: p>0.05, Two-Way-ANOVA, depicted is mean+SD).

Mentions: We then asked whether virus specificity, namely infection with MCMV, is necessary to activate FL from the replicon vector. As the core replication machinery is conserved in herpesviruses, we determined whether a herpesvirus from another subfamily could induce gene expression by the MCMV oriLyt system. As NIH3T3 cells are also permissive for the MHV68 γ-herpesvirus, which induces lytic replication in almost all infected mouse cells, we examined infection of an isolated cell clone, luc-ori cl.1, with MHV68 and MCMV (Fig. 2A). Whereas infection with MCMV resulted in typically high induction of FL expression (by three orders of magnitude), infection with MHV68 resulted only in a marginal increase of (∼5–10-fold). Because only MCMV was able to fully activate expression of the MCMV-oriLyt-replicon, the induction of the replicon system appears to be specific for infection with the corresponding herpesvirus.


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)

Induction of replicon vector is dependent on the MCMV DNA replication.(A) Induction of MCMV oriLyt is specific to MCMV infection. Cell line luc-ori cl.1 was infected with MCMV (beta herpesvirus; MOI = 0.5) or murine herpesvirus 68 (MHV68, gamma herpesvirus; MOI = 0.5) or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (B) NIH3T3 cells were stably transfected with pEpibo-luc (luc t1) or pEpibo-luc-ori (luc-ori t3). Depicted is the ratio of FL expression of the resulting cell pools before and after infection with MCMV at an MOI of 1. In the luc t1 cell pool, lacking the oriLyt sequence, FL expression is not enhanced by infection, in contrast to the luc-ori t3 cell pool, in which FL expression is induced about 40 fold at 36 h p.i. (C) NIH3T3 or luc-ori cell lines (luc-ori cl.1–cl.4) were infected with MCMV at an MOI of 0.5 (hatched bars) or left uninfected (plain bars). In addition, cell lines were either treated with phosponoacidic acid (PAA, 300 µg/ml, black bars) or left untreated (white bars). 36 h p.i. FL induction was measured via bioluminescence assay. While reporter expression is induced up to 1000-fold in MCMV infected cells lines cl.1–3, inhibition of viral DNA polymerase by PAA blocks the induction of FL expression. (D) Induction can be inhibited with PAA completely if it is added before replication has started and can be reduced if added after the onset of DNA replication. Cell line luc-ori cl.1 was infected with MCMV at an MOI = 0.5 or left uninfected. Arrows indicate time points when 300 µg/ml PAA was added to the cells. Cells were harvested at indicated time points and FL was measured via bioluminescence assay. Induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (p.i., post infection; BG = Background; ***: p<0.001, ns: p>0.05, Two-Way-ANOVA, depicted is mean+SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369935&req=5

ppat-1002728-g002: Induction of replicon vector is dependent on the MCMV DNA replication.(A) Induction of MCMV oriLyt is specific to MCMV infection. Cell line luc-ori cl.1 was infected with MCMV (beta herpesvirus; MOI = 0.5) or murine herpesvirus 68 (MHV68, gamma herpesvirus; MOI = 0.5) or left untreated. 36 h p.i. a bioluminescence assay was performed and the induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (B) NIH3T3 cells were stably transfected with pEpibo-luc (luc t1) or pEpibo-luc-ori (luc-ori t3). Depicted is the ratio of FL expression of the resulting cell pools before and after infection with MCMV at an MOI of 1. In the luc t1 cell pool, lacking the oriLyt sequence, FL expression is not enhanced by infection, in contrast to the luc-ori t3 cell pool, in which FL expression is induced about 40 fold at 36 h p.i. (C) NIH3T3 or luc-ori cell lines (luc-ori cl.1–cl.4) were infected with MCMV at an MOI of 0.5 (hatched bars) or left uninfected (plain bars). In addition, cell lines were either treated with phosponoacidic acid (PAA, 300 µg/ml, black bars) or left untreated (white bars). 36 h p.i. FL induction was measured via bioluminescence assay. While reporter expression is induced up to 1000-fold in MCMV infected cells lines cl.1–3, inhibition of viral DNA polymerase by PAA blocks the induction of FL expression. (D) Induction can be inhibited with PAA completely if it is added before replication has started and can be reduced if added after the onset of DNA replication. Cell line luc-ori cl.1 was infected with MCMV at an MOI = 0.5 or left uninfected. Arrows indicate time points when 300 µg/ml PAA was added to the cells. Cells were harvested at indicated time points and FL was measured via bioluminescence assay. Induction of the FL was calculated as the ratio of RLU of infected to uninfected cells. (p.i., post infection; BG = Background; ***: p<0.001, ns: p>0.05, Two-Way-ANOVA, depicted is mean+SD).
Mentions: We then asked whether virus specificity, namely infection with MCMV, is necessary to activate FL from the replicon vector. As the core replication machinery is conserved in herpesviruses, we determined whether a herpesvirus from another subfamily could induce gene expression by the MCMV oriLyt system. As NIH3T3 cells are also permissive for the MHV68 γ-herpesvirus, which induces lytic replication in almost all infected mouse cells, we examined infection of an isolated cell clone, luc-ori cl.1, with MHV68 and MCMV (Fig. 2A). Whereas infection with MCMV resulted in typically high induction of FL expression (by three orders of magnitude), infection with MHV68 resulted only in a marginal increase of (∼5–10-fold). Because only MCMV was able to fully activate expression of the MCMV-oriLyt-replicon, the induction of the replicon system appears to be specific for infection with the corresponding herpesvirus.

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