Testing measurement of antiviral agents.

<p>HFF infected in 96-well plates and fluorescence was measured in live cells by fluorescence spectrometry or detected by live microscopy at 10-fold magnification. (<b>A</b>) The relationship of relative fluorescence intensity (RFI) and multiplicity of infection is shown in the left panel for cells infected with the indicated viruses for 8 days. The panels on the right show cells infected with TB4-IE2-EYFP (5 dpi) and TB4-UL83-EYFP or TB4-UL32-EYFP (8 dpi) at the indicated MOI. (<b>B</b>) Measurement of neutralizing activity using TB4-IE2-EYFP. Virus was incubated with different dilutions of Flebogamma^R (5%) (upper row) or supernatant from hybridoma cell lines producing antibodies directed against anti-gH (14-4B) (middle row) or anti-gB (27–39) (bottom row). HFF were infected at MOI 0.1 and relative fluorescence intensities recorded at 9 dpi are shown in the panels on the left. Corresponding microscopic images are shown in the panels on the right.</p

Immunofluorescence analysis of HFF cells infected with recombinant viruses.

<p>The co-localisation of viral protein and EYFP in HFF infected with the recombinant virus was detected by fluorescence microscopy at 40× magnification. HFF were infected with TB4-IE2-EYFP (<b>A</b>), TB4-UL83-EYFP (<b>B</b>) or TB4-UL32-EYFP (<b>C</b>) with MOI 1 and fixated at 6, 24, 48 and 72 hpi as indicated on the left side. Cells were stained with mouse monoclonal antibodies directed against IE1–2 (E13) (A), ppUL83 (B) or ppUL32 (XP-1) (C) and Alexa-Fluor555 conjugated secondary antibody. Single channel recordings of DNA (Dapi), EYFP and the viral protein portion (red staining with Alexa-Fluor555) are shown in the left three columns; merged images are in the rightmost column. The time after infection is indicated on the left side.</p

Western blot analysis of recombinant viruses.

<p>At 72 hpi, TB4- and recombinant virus-infected HFF (MOI of 1) were harvested, lysed and the proteins were separated by a 10% SDS-PAGE. Detection was done by using specific antibodies against the viral fusion partner or the fluorescent protein. (<b>A</b>) detection of IE1+2 with polyclonal rabbit anti-IE1+2. (<b>B</b>) anti-GFP antibody. (<b>C</b>) detection of ppUL83 and ppUL83-EYFP with specific anti-pp65 or (<b>D</b>) anti-GFP antibody. ppUL32 was detected with an specific anti-ppUL32 antibody (<b>E</b>) or also with the anti-GFP antibody (<b>F</b>). In (<b>G</b>) the lysates of cell free viral stocks of TB4-wt (lane 1), TB4-IE2-EYFP (lane 2), TB4-UL83-EYFP (lane 3) and TB4-UL32-EYFP (lane 4) were separated, blotted and detected with antibodies specific for major capsid protein (MCP, pUL86), GFP, ppUL32, ppUL82 and ppUL83. The stocks had been adjusted to equal levels of MCP.</p

Demonstration of the genomic rearrangement by Southern blot analysis.

<p>Respective recombinant BAC DNAs were digested with EcoRV and separated by agarose gel electrophoresis (A), (D) and (G). A size marker is shown on the gel with the fragment sizes indicated on the right side. Subsequently the gel was blotted and subjected to southern hybridization analysis using the [³²P]dCTP labelled gene-specific (B, E and H) detailed below or an EYFP-specific probe (C, F and I). (<b>A</b>) BAC DNA of HCMV TB4-IE2-EYFP-kana⁺ (lane 1), TB4-IE2-EYFP-kana⁻ (lane 2) and TB4wt (lane 3). (<b>B</b>) Southern analysis using UL123 (IE2)- and UL120-specific probes. (<b>D</b>) BAC DNA of HCMV TB4-UL83-EYFP-kana⁺ (lane 1), TB4-UL83-EYFP-kana⁻ (lane 2) and TB4wt (lane 3). (<b>E</b>) Southern blot using UL83- and UL82-specific probes. (<b>G</b>) BAC DNA of HCMV TB4-UL32-EYFP-kana⁺ (lane 1), TB4 UL32-EYFP-kana⁻ (lane 2) and TB4wt (lane 3). (<b>H</b>) Southern analysis using UL32- and UL31-specific probes.</p

Screening a kinase inhibitor library.

<p>A kinase inhibitor library (lanes 1–80) was used to measure cell viability (A) or inhibition of virus replication using TB4-IE2-EYFP (B), TB4-UL83-EYFP (C) or TB4-UL32-EYFP (D). All measurements were done at 4 dpi. (A) To determine cell viability all cells remained uninfected. Cell viability of mock infected cells was set 1.0. (B)–(D) Fluorescence intensity was measured in a Tecan Safire 2. Background (uninfected cells) was subtracted from all samples and normalized to infected cells (100%). Inhibitors were ordered according to the inhibition of TB4-IE2-EYFP.</p

High content analysis of nucleocytoplasmic shuttling of ppUL83-EYFP.

<p>Human foreskin fibroblasts were infected with TB4-UL83-EYFP and treated with different concentrations of ganciclovir (A) or kinase inhibitors (B). Fluorescence images were recorded and analyzed in a BD Pathway 855 for nuclear or cytoplasmic localization. (A) In the diagram on the left the ratio of cells with a cytoplasmic/nuclear staining of higher than 0.7 is shown. Infected cell were treated with 100 µM (3), 33 µM (4), 11 µM (5), 3.7 µM (6) or 1.2 µM (7) ganciclovir. Uninfected (1) or infected (2) cells were used as controls. The localization of ppUL83-EYFP in selected samples is shown in the right panel; numbers indicate the respective sample of diagram. (B) The diagram on the top shows the ratio of cells with a cytoplasmic/nuclear staining of higher than 0.7, as above. Kinase inhibitors (1–80) are ordered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009174#pone-0009174-g007" target="_blank">Fig. 7</a>. Leptomycin B (81) was used as positive control. The localization of ppUL83-EYFP in selected samples is shown in the lower panels. Several samples were marked as not applicable (na), since too few cells were above an intensity threshold for EYFP fluorescence.</p

Virus growth kinetics of recombinant viruses in human foreskin fibroblasts.

<p>HFF were infected with TB4-wt (circles), TB4-IE2-EYFP (squares), TB4-UL83-EYFP (triangles) and TB4-UL32-EYFP (crosses) at MOI of 1 (<b>A</b>), 0.1 (<b>B</b>) and 0.01 (<b>C</b>). Supernatant was collected and titrated over a range of 10 days post infection.</p

Surface expression of CD14, CD163 and CD206 on untreated THP-1 cells (mock) or after differentiation into macrophage-like cells by stimulation with PMA alone (PMA) or in combination with 50 ng ml⁻¹ M-CSF (M+PMA) or GM-CSF (GM+PMA).

<p>All experiments were performed in triplicates (n = 3). Histogram plots show surface marker staining as hatched areas and isotype controls as black lines.</p

Fluorescence microscopy of GM-Mφ (GM) and M-Mφ (M) generated from monocytes isolated by negative (neg.) or positive (pos.) selection and infected with <i>Lm</i> at an MOI of 10 (A).

<p>Nuclei of macrophages are stained with DAPI (blue) and <i>Lm</i> was stained with a specific antibody (red). Scale bar is 10 µm. Infection of GM-Mφ (-GM or +GM, black bars) and M-Mφ (-M or +M, white bars) with <i>Lm</i> was determined at different multiplicities of infection (MOI) and fluorescence microscopy images were analysed either for the percentage of infected cells (B) or the number of bacteria per infected cell (C). For each MOI and macrophage phenotype, infected cells and <i>Lm</i> per infected cell were counted in random microscopic fields of view of three donors. For each donor, three independent fields of view with at least 100 cells were analysed. Statistical analysis was performed on the means of different donors by pairwise comparison of GM-Mφ vs. M-Mφ at the different MOIs using Student's <i>t</i>-test (n.s.: not significant).</p

Phagocytosis of fluorescent latex beads by GM-Mφ and M-Mφ generated from monocytes isolated by negative (neg.) or positive (pos.) selection as measured by flow cytometry.

<p>Histogram plots show macrophages incubated with latex beads (hatched area) and cells alone (black lines) as control. Percentage of macrophages which are positve for latex beads are indicated in each histogram plot. Results from one representative donor are shown and similar results were obtained with cells of at least three different donors.</p