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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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OriLyt dependent transgene expression in vivo.Generation of virus inducible oriLyt dependent luciferase animal (VIOLA). (A) (1.) mES cell clones were transfected with pEpibo-luc-ori and (2.) pre-selected for their induction capacity by MCMV infection in vitro. (3.) Mouse lines were analyzed for expression of FL before and after infection in explant cultures of different organs. (B) mES cells were transfected with pEpibo-luc-ori and 8 clones were isolated. A portion of the clones were partially differentiated for 3 weeks and then infected with MCMV at an MOI of 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. mES clones A3 and B8 were selected for generation of transgenic animals. (C) Explant cultures of lungs taken from mice of the Viola–A strain, backcrossed one (2nd generation) or two (3rd generation) times to 129X1/SvJ, were produced and were infected with MCMV-wt with an MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). As a control mice of the background strain 129X1/SvJ (wt/wt) were used. (D) Explant cultures from bone marrow (BM), heart (He), muscle (Mu), fat (FA), spleen (Sp) and salivary glands (Sg) were grown from a VIOLA-A mouse of the fourth generation. Cells were infected with MCMV-wt with MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). (p.i. = post infection ; BG = background of luciferin).
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ppat-1002728-g006: OriLyt dependent transgene expression in vivo.Generation of virus inducible oriLyt dependent luciferase animal (VIOLA). (A) (1.) mES cell clones were transfected with pEpibo-luc-ori and (2.) pre-selected for their induction capacity by MCMV infection in vitro. (3.) Mouse lines were analyzed for expression of FL before and after infection in explant cultures of different organs. (B) mES cells were transfected with pEpibo-luc-ori and 8 clones were isolated. A portion of the clones were partially differentiated for 3 weeks and then infected with MCMV at an MOI of 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. mES clones A3 and B8 were selected for generation of transgenic animals. (C) Explant cultures of lungs taken from mice of the Viola–A strain, backcrossed one (2nd generation) or two (3rd generation) times to 129X1/SvJ, were produced and were infected with MCMV-wt with an MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). As a control mice of the background strain 129X1/SvJ (wt/wt) were used. (D) Explant cultures from bone marrow (BM), heart (He), muscle (Mu), fat (FA), spleen (Sp) and salivary glands (Sg) were grown from a VIOLA-A mouse of the fourth generation. Cells were infected with MCMV-wt with MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). (p.i. = post infection ; BG = background of luciferin).

Mentions: As already observed for several attenuated MCMV-mutants, the reduction in virus titer of 99% observed in tissue culture may result in even stronger attenuation if the system is operative in vivo[44]. To test the oriLyt system in vivo, we transferred the conditional DN principle to the natural host of MCMV, the mouse. To our knowledge, no transgenic mouse has been created based on the pEPI-1 vector. Therefore, to assess episomal stability and to test de-silencing in vivo, we generated transgenic mice carrying the pEpibo-luc-ori construct. This should permit the general concept to be studied in vivo (Fig. 6A). To this end, we transfected murine embryonic stem cells (mES line E14) with pEpibo-luc-ori and isolated eight cell clones. Note that mES cells are non-permissive for MCMV infection. To identify cell clones suitable for blastocyst injection, several cell populations were differentiated for 3 weeks to enable productive MCMV infection [45]. To study virus specific de-silencing in differentiated mES, the bioluminescence signal was measured both prior to and after MCMV infection. In three of the eight clones, no FL expression was detected under any conditions (A10, B1, B9); three other clones showed a weak increase in FL expression after infection (A2, A6, B11) and two clones were induced 7- (A3) and 30-fold (B8) (Fig. 6B).


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)

OriLyt dependent transgene expression in vivo.Generation of virus inducible oriLyt dependent luciferase animal (VIOLA). (A) (1.) mES cell clones were transfected with pEpibo-luc-ori and (2.) pre-selected for their induction capacity by MCMV infection in vitro. (3.) Mouse lines were analyzed for expression of FL before and after infection in explant cultures of different organs. (B) mES cells were transfected with pEpibo-luc-ori and 8 clones were isolated. A portion of the clones were partially differentiated for 3 weeks and then infected with MCMV at an MOI of 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. mES clones A3 and B8 were selected for generation of transgenic animals. (C) Explant cultures of lungs taken from mice of the Viola–A strain, backcrossed one (2nd generation) or two (3rd generation) times to 129X1/SvJ, were produced and were infected with MCMV-wt with an MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). As a control mice of the background strain 129X1/SvJ (wt/wt) were used. (D) Explant cultures from bone marrow (BM), heart (He), muscle (Mu), fat (FA), spleen (Sp) and salivary glands (Sg) were grown from a VIOLA-A mouse of the fourth generation. Cells were infected with MCMV-wt with MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). (p.i. = post infection ; BG = background of luciferin).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002728-g006: OriLyt dependent transgene expression in vivo.Generation of virus inducible oriLyt dependent luciferase animal (VIOLA). (A) (1.) mES cell clones were transfected with pEpibo-luc-ori and (2.) pre-selected for their induction capacity by MCMV infection in vitro. (3.) Mouse lines were analyzed for expression of FL before and after infection in explant cultures of different organs. (B) mES cells were transfected with pEpibo-luc-ori and 8 clones were isolated. A portion of the clones were partially differentiated for 3 weeks and then infected with MCMV at an MOI of 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. mES clones A3 and B8 were selected for generation of transgenic animals. (C) Explant cultures of lungs taken from mice of the Viola–A strain, backcrossed one (2nd generation) or two (3rd generation) times to 129X1/SvJ, were produced and were infected with MCMV-wt with an MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). As a control mice of the background strain 129X1/SvJ (wt/wt) were used. (D) Explant cultures from bone marrow (BM), heart (He), muscle (Mu), fat (FA), spleen (Sp) and salivary glands (Sg) were grown from a VIOLA-A mouse of the fourth generation. Cells were infected with MCMV-wt with MOI of 0.5 (hatched bars) or left untreated (white bars). 24 h p.i. FL expression was determined by measuring relative light units (RLU). (p.i. = post infection ; BG = background of luciferin).
Mentions: As already observed for several attenuated MCMV-mutants, the reduction in virus titer of 99% observed in tissue culture may result in even stronger attenuation if the system is operative in vivo[44]. To test the oriLyt system in vivo, we transferred the conditional DN principle to the natural host of MCMV, the mouse. To our knowledge, no transgenic mouse has been created based on the pEPI-1 vector. Therefore, to assess episomal stability and to test de-silencing in vivo, we generated transgenic mice carrying the pEpibo-luc-ori construct. This should permit the general concept to be studied in vivo (Fig. 6A). To this end, we transfected murine embryonic stem cells (mES line E14) with pEpibo-luc-ori and isolated eight cell clones. Note that mES cells are non-permissive for MCMV infection. To identify cell clones suitable for blastocyst injection, several cell populations were differentiated for 3 weeks to enable productive MCMV infection [45]. To study virus specific de-silencing in differentiated mES, the bioluminescence signal was measured both prior to and after MCMV infection. In three of the eight clones, no FL expression was detected under any conditions (A10, B1, B9); three other clones showed a weak increase in FL expression after infection (A2, A6, B11) and two clones were induced 7- (A3) and 30-fold (B8) (Fig. 6B).

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