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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 a dominant-negative protein by the replicon vector inhibits viral growth.(A) In the transgenic cell line NIH3T3: gfpscp-ori (gfpscp-ori), infection with MCMV induces replication of the construct and thereby activates the production of the dominant-negative protein GFPSCP (green symbols). This protein blocks egress from the nucleus and viral spread is inhibited. (B) NIH3T3 (white bars) and gfpscp-ori (black bars) cell lines were infected with MCMV-wt at MOI of 0.1. At the indicated time points, infectious virus was quantified in the supernatants by standard plaque assay. Due to the production of the inhibitory protein, the cell line gfpscp-ori releases 100–200 - fold fewer viruses into supernatants. (C) NIH3T3 or gfpscp-ori cell lines were infected with MCMV-mCherry at an MOI of 0.1 and expression of GFPSCP and mCherry was assessed 5 days p.i. by fluorescence microscopy. The mCherry protein is expressed with late kinetics and serves as an infection marker. Only in infected cells GFPSCP is produced and localizes according to its typical pattern in nuclear speckles. Plaques on the gfpscp-ori cell line are reduced in size. (D) FISH of metaphase spreads of uninfected gfpscp-ori cells (4n = 76). Three different probes complementary to the gfpscp gene (green), bsr gene (pink) and oriLyt (red) were used. Probes co-localized to DAPI stained extrachromosomal spots, indicating an episomal persistence of pEpibo-gfpscp-ori.
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ppat-1002728-g005: Induction of a dominant-negative protein by the replicon vector inhibits viral growth.(A) In the transgenic cell line NIH3T3: gfpscp-ori (gfpscp-ori), infection with MCMV induces replication of the construct and thereby activates the production of the dominant-negative protein GFPSCP (green symbols). This protein blocks egress from the nucleus and viral spread is inhibited. (B) NIH3T3 (white bars) and gfpscp-ori (black bars) cell lines were infected with MCMV-wt at MOI of 0.1. At the indicated time points, infectious virus was quantified in the supernatants by standard plaque assay. Due to the production of the inhibitory protein, the cell line gfpscp-ori releases 100–200 - fold fewer viruses into supernatants. (C) NIH3T3 or gfpscp-ori cell lines were infected with MCMV-mCherry at an MOI of 0.1 and expression of GFPSCP and mCherry was assessed 5 days p.i. by fluorescence microscopy. The mCherry protein is expressed with late kinetics and serves as an infection marker. Only in infected cells GFPSCP is produced and localizes according to its typical pattern in nuclear speckles. Plaques on the gfpscp-ori cell line are reduced in size. (D) FISH of metaphase spreads of uninfected gfpscp-ori cells (4n = 76). Three different probes complementary to the gfpscp gene (green), bsr gene (pink) and oriLyt (red) were used. Probes co-localized to DAPI stained extrachromosomal spots, indicating an episomal persistence of pEpibo-gfpscp-ori.

Mentions: Next, we studied whether virus replication can be inhibited by a DN transgene induced late in the viral cascade. Inhibition of viral spread by the use of DN viral proteins, called intracellular immunization, was proposed by Baltimore in 1988 [36] and was inspired by the work of Friedman and colleagues [37]. They provided the proof-of-principle experiment showing that a truncated VP16 protein could reduce the replication of HSV-1 when stably expressed by the host cell line. Since then, toxicity caused by DN transgenes has been a problem [37]–[40]. To test the usefulness of our system for studying dominant-negative effects, we cloned the DN gfpscp gene [41], [42], which codes for a fusion between GFP and the small capsid protein (SCP) of MCMV, into the oriLyt vector. During infection of the NIH3T3:gfpscp-ori (gfpscp-ori) cell line, MCMV should de-silence expression of the inhibitory protein GFPSCP. Strong expression of GFPSCP should, in turn, block the egress of viral capsids from the nucleus (Fig. 5A).


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 a dominant-negative protein by the replicon vector inhibits viral growth.(A) In the transgenic cell line NIH3T3: gfpscp-ori (gfpscp-ori), infection with MCMV induces replication of the construct and thereby activates the production of the dominant-negative protein GFPSCP (green symbols). This protein blocks egress from the nucleus and viral spread is inhibited. (B) NIH3T3 (white bars) and gfpscp-ori (black bars) cell lines were infected with MCMV-wt at MOI of 0.1. At the indicated time points, infectious virus was quantified in the supernatants by standard plaque assay. Due to the production of the inhibitory protein, the cell line gfpscp-ori releases 100–200 - fold fewer viruses into supernatants. (C) NIH3T3 or gfpscp-ori cell lines were infected with MCMV-mCherry at an MOI of 0.1 and expression of GFPSCP and mCherry was assessed 5 days p.i. by fluorescence microscopy. The mCherry protein is expressed with late kinetics and serves as an infection marker. Only in infected cells GFPSCP is produced and localizes according to its typical pattern in nuclear speckles. Plaques on the gfpscp-ori cell line are reduced in size. (D) FISH of metaphase spreads of uninfected gfpscp-ori cells (4n = 76). Three different probes complementary to the gfpscp gene (green), bsr gene (pink) and oriLyt (red) were used. Probes co-localized to DAPI stained extrachromosomal spots, indicating an episomal persistence of pEpibo-gfpscp-ori.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002728-g005: Induction of a dominant-negative protein by the replicon vector inhibits viral growth.(A) In the transgenic cell line NIH3T3: gfpscp-ori (gfpscp-ori), infection with MCMV induces replication of the construct and thereby activates the production of the dominant-negative protein GFPSCP (green symbols). This protein blocks egress from the nucleus and viral spread is inhibited. (B) NIH3T3 (white bars) and gfpscp-ori (black bars) cell lines were infected with MCMV-wt at MOI of 0.1. At the indicated time points, infectious virus was quantified in the supernatants by standard plaque assay. Due to the production of the inhibitory protein, the cell line gfpscp-ori releases 100–200 - fold fewer viruses into supernatants. (C) NIH3T3 or gfpscp-ori cell lines were infected with MCMV-mCherry at an MOI of 0.1 and expression of GFPSCP and mCherry was assessed 5 days p.i. by fluorescence microscopy. The mCherry protein is expressed with late kinetics and serves as an infection marker. Only in infected cells GFPSCP is produced and localizes according to its typical pattern in nuclear speckles. Plaques on the gfpscp-ori cell line are reduced in size. (D) FISH of metaphase spreads of uninfected gfpscp-ori cells (4n = 76). Three different probes complementary to the gfpscp gene (green), bsr gene (pink) and oriLyt (red) were used. Probes co-localized to DAPI stained extrachromosomal spots, indicating an episomal persistence of pEpibo-gfpscp-ori.
Mentions: Next, we studied whether virus replication can be inhibited by a DN transgene induced late in the viral cascade. Inhibition of viral spread by the use of DN viral proteins, called intracellular immunization, was proposed by Baltimore in 1988 [36] and was inspired by the work of Friedman and colleagues [37]. They provided the proof-of-principle experiment showing that a truncated VP16 protein could reduce the replication of HSV-1 when stably expressed by the host cell line. Since then, toxicity caused by DN transgenes has been a problem [37]–[40]. To test the usefulness of our system for studying dominant-negative effects, we cloned the DN gfpscp gene [41], [42], which codes for a fusion between GFP and the small capsid protein (SCP) of MCMV, into the oriLyt vector. During infection of the NIH3T3:gfpscp-ori (gfpscp-ori) cell line, MCMV should de-silence expression of the inhibitory protein GFPSCP. Strong expression of GFPSCP should, in turn, block the egress of viral capsids from the nucleus (Fig. 5A).

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