6 research outputs found
Induction of a dominant-negative protein by the replicon vector inhibits viral growth.
<p>(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 <i>gfpscp</i> gene (green), <i>bsr</i> gene (pink) and oriLyt (red) were used. Probes co-localized to DAPI stained extrachromosomal spots, indicating an episomal persistence of pEpibo-gfpscp-ori.</p
Infection with MCMV reactivates silenced replicon vector encoded reporter gene expression.
<p>(A) In two stable NIH3T3 cell pools transfected with pEpibo-luc-ori (luc-ori t1, luc-ori t2) expression of FL was measured in absence of infection (plain bars, - MCMV) at the indicated weeks after transfection. Reporter gene expression is lost in uninfected cells over time. At the same time points the pools were infected with MCMV at an MOI of 0.5 (hatched bars, +MCMV). 24 h p.i. of both luc-ori t1 and t2 with high expression of FL was induced. NIH3T3 fibroblasts served as a control to determine background signal (BG) of the luciferin substrate. (B) Vector pEpibo-luc-ori was inactivated by histone deacetylation. Four cell clones (cl. 1–4) derived from subcloning of luc-ori t1 were subjected to treatment with 25 µM 5′ aza-cytidine (5′Aza, gray bars), an inhibitor of CpG-methylation, 330 nM Trichostatin A (TSA, black bars) for 36 h or left untreated (mock, open bars). FL expression was analyzed in comparison to parental NIH3T3 cells. FL expression was significantly enhanced by de-condensing histone packaging through TSA treatment (*** p<0.001, ns p>0.05, Two-Way-Anova, depicted is mean+SD). RLU (relative light units), p.i. post infection, weeks = weeks post transfection of pEpibo-luc-ori.</p
pEpibo-luc-ori is amplified upon MCMV infection.
<p>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 <i>bsr</i> coding sequence compared to the cellular gene lamin B receptor (<i>lbr</i>).</p
Replicon vector based trans-complementation.
<p>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.</p
OriLyt dependent transgene expression in vivo.
<p>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 <i>in vitro</i>. (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 (2<sup>nd</sup> generation) or two (3<sup>rd</sup> 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).</p
Induction of replicon vector is dependent on the MCMV DNA replication.
<p>(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).</p