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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

Replicon vector based trans-complementation.A–C trans-complementation of a glycoprotein. (A) Trans-complementation of the late glycoprotein gO can be facilitated by oriLyt-induced gene expression. NIH3T3 (striped bars) or gO-ori (plain bars) cell lines have been infected with MCMV-wt (white bars) or MCMVΔgO (black bars) at an MOI = 0.05 and centrifugal enhancement. At the indicated time points the number of the infectious virus was quantified in the culture supernatants by standard plaque assay. (B) Immunofluorescence microscopy of infected NIH3T3 or gO-ori cells performed 5 days post infection (MOI = 0.05). Infected cells were stained with the anti-IE antibody CHROMA-101. While MCMVΔgO is restricted to focal cell-to-cell spread in NIH3T3 fibroblasts, it spreads like wild type in the trans-complementing cell line gO-ori. (C) PCR analysis of virus progeny produced on gO-ori cell line. Viral DNA was isolated from supernatants of the viral growth curve of (A) from day 5, cleared from residual cells, and analyzed by PCR. The virus polymerase gene, M54 served as a positive control for viral infection. The cellular gene lbr, served as a control for the lack of residual genomic DNA. PCR on the m74 gene, encoding gO, showed the presence of the gene in MCMV-wt and the lack of it in MCMVΔgO. No uptake and recombination of gO after passage over gO-ori cells could be detected. D–E Trans-complementation of M50, a protein essential for nuclear export of viral capsids. (D) Detection of the M50HA protein (∼35 kDa) in cell lysates of NIH3T3, M50-ori t1 and M50-ori t2. The respective cell lines were infected with MCMV-wt at an MOI of 1 and cell lysates harvested 36 h p.i. (E) Growth of MCMVΔM50-cherry on complementing and non-complementing cell line. Supernatant of the reconstitution of MCMVΔM50-cherry on M50-ori cl.2.1 was serial diluted and used to infect NIH3T3 or M50-ori cells. The trans-complemented virus MCMVΔM50-cherry/M50HA could spread in M50-ori cells, but produced only primary infection in NIH3T3.
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ppat-1002728-g004: Replicon vector based trans-complementation.A–C trans-complementation of a glycoprotein. (A) Trans-complementation of the late glycoprotein gO can be facilitated by oriLyt-induced gene expression. NIH3T3 (striped bars) or gO-ori (plain bars) cell lines have been infected with MCMV-wt (white bars) or MCMVΔgO (black bars) at an MOI = 0.05 and centrifugal enhancement. At the indicated time points the number of the infectious virus was quantified in the culture supernatants by standard plaque assay. (B) Immunofluorescence microscopy of infected NIH3T3 or gO-ori cells performed 5 days post infection (MOI = 0.05). Infected cells were stained with the anti-IE antibody CHROMA-101. While MCMVΔgO is restricted to focal cell-to-cell spread in NIH3T3 fibroblasts, it spreads like wild type in the trans-complementing cell line gO-ori. (C) PCR analysis of virus progeny produced on gO-ori cell line. Viral DNA was isolated from supernatants of the viral growth curve of (A) from day 5, cleared from residual cells, and analyzed by PCR. The virus polymerase gene, M54 served as a positive control for viral infection. The cellular gene lbr, served as a control for the lack of residual genomic DNA. PCR on the m74 gene, encoding gO, showed the presence of the gene in MCMV-wt and the lack of it in MCMVΔgO. No uptake and recombination of gO after passage over gO-ori cells could be detected. D–E Trans-complementation of M50, a protein essential for nuclear export of viral capsids. (D) Detection of the M50HA protein (∼35 kDa) in cell lysates of NIH3T3, M50-ori t1 and M50-ori t2. The respective cell lines were infected with MCMV-wt at an MOI of 1 and cell lysates harvested 36 h p.i. (E) Growth of MCMVΔM50-cherry on complementing and non-complementing cell line. Supernatant of the reconstitution of MCMVΔM50-cherry on M50-ori cl.2.1 was serial diluted and used to infect NIH3T3 or M50-ori cells. The trans-complemented virus MCMVΔM50-cherry/M50HA could spread in M50-ori cells, but produced only primary infection in NIH3T3.

Mentions: We cloned the m74 gene, coding gO [35], into the oriLyt vector to generate the responding cell line NIH3T3:gO-ori (gO-ori). MCMV, like HCMV, lacking gO is restricted to focal spreading and the release of infectious virions into the supernatant is hampered [35]. In the cell line containing the gO-ori vector, however, infection with MCMV-ΔgO should lead to pEpibo-gO-ori replication and m74 gene expression; thereby reconstituting release of infectious progeny. MCMV-ΔgO released from NIH3T3 about ∼2–2.5 orders of magnitude less virus than MCMV-wt. By contrast, growth in the complementing cell line resulted in comparable titers for both MCMV-ΔgO and MCMV-wt (Fig. 4A). Thus, the virus defect was rescued and the virus was no longer restricted to a focal growth pattern (Fig. 4B). This confirms that the replicon expression system can be used to efficiently produce late viral proteins in trans. To determine whether the viral genome reverts to a wt-like virus due to recombination with the episomal replicon vector, PCR analysis of the m74 gene (gO) was performed using viruses harvested on day 5 from the supernatants used in the growth curve experiments. Supernatants were centrifuged to remove cells and the cellular DNA was digested with DNase to discriminate between the m74 gene in the replicon vectors in the cells and the viral genome. The fact that the supernatants were free from cellular debris was confirmed by the lack of the cellular gene lbr. Presence of viral genomic DNA was confirmed by amplifying the viral DNA polymerase, M54. Whereas all supernatants were positive for the M54 gene and negative for the lbr gene, m74 was detected only in MCMV-wt, and not in MCMVΔgO, regardless of whether it originated from NIH3T3 or gO-ori cells (Fig. 4C). Furthermore, we never observed any reversion of the phenotypic restriction of MCMVΔgO to cell-to-cell spreading (data not shown). No recombination was detected between the replicon vector and the MCMVΔgO virus.


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)

Replicon vector based trans-complementation.A–C trans-complementation of a glycoprotein. (A) Trans-complementation of the late glycoprotein gO can be facilitated by oriLyt-induced gene expression. NIH3T3 (striped bars) or gO-ori (plain bars) cell lines have been infected with MCMV-wt (white bars) or MCMVΔgO (black bars) at an MOI = 0.05 and centrifugal enhancement. At the indicated time points the number of the infectious virus was quantified in the culture supernatants by standard plaque assay. (B) Immunofluorescence microscopy of infected NIH3T3 or gO-ori cells performed 5 days post infection (MOI = 0.05). Infected cells were stained with the anti-IE antibody CHROMA-101. While MCMVΔgO is restricted to focal cell-to-cell spread in NIH3T3 fibroblasts, it spreads like wild type in the trans-complementing cell line gO-ori. (C) PCR analysis of virus progeny produced on gO-ori cell line. Viral DNA was isolated from supernatants of the viral growth curve of (A) from day 5, cleared from residual cells, and analyzed by PCR. The virus polymerase gene, M54 served as a positive control for viral infection. The cellular gene lbr, served as a control for the lack of residual genomic DNA. PCR on the m74 gene, encoding gO, showed the presence of the gene in MCMV-wt and the lack of it in MCMVΔgO. No uptake and recombination of gO after passage over gO-ori cells could be detected. D–E Trans-complementation of M50, a protein essential for nuclear export of viral capsids. (D) Detection of the M50HA protein (∼35 kDa) in cell lysates of NIH3T3, M50-ori t1 and M50-ori t2. The respective cell lines were infected with MCMV-wt at an MOI of 1 and cell lysates harvested 36 h p.i. (E) Growth of MCMVΔM50-cherry on complementing and non-complementing cell line. Supernatant of the reconstitution of MCMVΔM50-cherry on M50-ori cl.2.1 was serial diluted and used to infect NIH3T3 or M50-ori cells. The trans-complemented virus MCMVΔM50-cherry/M50HA could spread in M50-ori cells, but produced only primary infection in NIH3T3.
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Related In: Results  -  Collection

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

ppat-1002728-g004: Replicon vector based trans-complementation.A–C trans-complementation of a glycoprotein. (A) Trans-complementation of the late glycoprotein gO can be facilitated by oriLyt-induced gene expression. NIH3T3 (striped bars) or gO-ori (plain bars) cell lines have been infected with MCMV-wt (white bars) or MCMVΔgO (black bars) at an MOI = 0.05 and centrifugal enhancement. At the indicated time points the number of the infectious virus was quantified in the culture supernatants by standard plaque assay. (B) Immunofluorescence microscopy of infected NIH3T3 or gO-ori cells performed 5 days post infection (MOI = 0.05). Infected cells were stained with the anti-IE antibody CHROMA-101. While MCMVΔgO is restricted to focal cell-to-cell spread in NIH3T3 fibroblasts, it spreads like wild type in the trans-complementing cell line gO-ori. (C) PCR analysis of virus progeny produced on gO-ori cell line. Viral DNA was isolated from supernatants of the viral growth curve of (A) from day 5, cleared from residual cells, and analyzed by PCR. The virus polymerase gene, M54 served as a positive control for viral infection. The cellular gene lbr, served as a control for the lack of residual genomic DNA. PCR on the m74 gene, encoding gO, showed the presence of the gene in MCMV-wt and the lack of it in MCMVΔgO. No uptake and recombination of gO after passage over gO-ori cells could be detected. D–E Trans-complementation of M50, a protein essential for nuclear export of viral capsids. (D) Detection of the M50HA protein (∼35 kDa) in cell lysates of NIH3T3, M50-ori t1 and M50-ori t2. The respective cell lines were infected with MCMV-wt at an MOI of 1 and cell lysates harvested 36 h p.i. (E) Growth of MCMVΔM50-cherry on complementing and non-complementing cell line. Supernatant of the reconstitution of MCMVΔM50-cherry on M50-ori cl.2.1 was serial diluted and used to infect NIH3T3 or M50-ori cells. The trans-complemented virus MCMVΔM50-cherry/M50HA could spread in M50-ori cells, but produced only primary infection in NIH3T3.
Mentions: We cloned the m74 gene, coding gO [35], into the oriLyt vector to generate the responding cell line NIH3T3:gO-ori (gO-ori). MCMV, like HCMV, lacking gO is restricted to focal spreading and the release of infectious virions into the supernatant is hampered [35]. In the cell line containing the gO-ori vector, however, infection with MCMV-ΔgO should lead to pEpibo-gO-ori replication and m74 gene expression; thereby reconstituting release of infectious progeny. MCMV-ΔgO released from NIH3T3 about ∼2–2.5 orders of magnitude less virus than MCMV-wt. By contrast, growth in the complementing cell line resulted in comparable titers for both MCMV-ΔgO and MCMV-wt (Fig. 4A). Thus, the virus defect was rescued and the virus was no longer restricted to a focal growth pattern (Fig. 4B). This confirms that the replicon expression system can be used to efficiently produce late viral proteins in trans. To determine whether the viral genome reverts to a wt-like virus due to recombination with the episomal replicon vector, PCR analysis of the m74 gene (gO) was performed using viruses harvested on day 5 from the supernatants used in the growth curve experiments. Supernatants were centrifuged to remove cells and the cellular DNA was digested with DNase to discriminate between the m74 gene in the replicon vectors in the cells and the viral genome. The fact that the supernatants were free from cellular debris was confirmed by the lack of the cellular gene lbr. Presence of viral genomic DNA was confirmed by amplifying the viral DNA polymerase, M54. Whereas all supernatants were positive for the M54 gene and negative for the lbr gene, m74 was detected only in MCMV-wt, and not in MCMVΔgO, regardless of whether it originated from NIH3T3 or gO-ori cells (Fig. 4C). Furthermore, we never observed any reversion of the phenotypic restriction of MCMVΔgO to cell-to-cell spreading (data not shown). No recombination was detected between the replicon vector and the MCMVΔgO virus.

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