Nanoparticle-Loaded Magnetophoretic Vesicles

Magnetic nanoparticles have been assembled into the bilayer membrane of block copolymer vesicles. The nanoparticles decorate the hydrophobic/hydrophilic interface, which leads to bridging of adjacent bilayers and the formation of oligo-lamellar vesicles. The nanoparticle uptake of the vesicles is sufficiently high to become magnetophoretic in external magnetic fields as shown by video microscopy

Construction of recombinant HCMV TB40E-EYFP.

<p>(<b>A</b>) Map of the pSL-FRT-EYFP plasmid. Open reading frames and FRT-sites are indicated. Primer binding sites used for amplification were gfpBAC-primer and gene-primer. (<b>B</b>) Scheme of ET-cloning. The EYFP/Kan-cassette was amplified by PCR and used for recET-based recombination to fuse EYFP to the C-terminus of the gene of interest. This resulted in a BAC still containing the kanamycin resistance gene (kan BAC). By transformation of a plasmid encoding the FLP recombinase the kanamycin resistance was removed, leaving a BAC with gene-EYFP fusion and a FRT-site (gene-YFP BAC).</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

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

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

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

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

Loss of NEMO degradation in autophagy-deficient MEFs, and MCMV-induced accumulation of LC3-II.

<p>(A) <i>atg5</i>^{−<i>/</i>−} and <i>atg5^+/+</i> MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting. (B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH₄Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.</p