Human Cytomegaloviruses Expressing Yellow Fluorescent Fusion Proteins - Characterization and Use in Antiviral Screening

Recombinant viruses labelled with fluorescent proteins are useful tools in molecular virology with multiple applications (e.g., studies on intracellular trafficking, protein localization, or gene activity). We generated by homologous recombination three recombinant cytomegaloviruses carrying the enhanced yellow fluorescent protein (EYFP) fused with the viral proteins IE-2, ppUL32 (pp150), and ppUL83 (pp65). In growth kinetics, the three viruses behaved all like wild type, even at low multiplicity of infection (MOI). The expression of all three fusion proteins was detected, and their respective localizations were the same as for the unmodified proteins in wild-type virus–infected cells. We established the in vivo measurement of fluorescence intensity and used the recombinant viruses to measure inhibition of viral replication by neutralizing antibodies or antiviral substances. The use of these viruses in a pilot screen based on fluorescence intensity and high-content analysis identified cellular kinase inhibitors that block viral replication. In summary, these viruses with individually EYFP-tagged proteins will be useful to study antiviral substances and the dynamics of viral infection in cell culture

The gene region UL128-UL131A of human cytomegalovirus (HCMV) is essential for monocyte infection and block of migration - Characterisation of the infection of primary human monocytes

Monocytes are targets of HCMV infection and vehicles for viral dissemination in vivo. By using two TB40E-derived BAC clones expressing the full-length (BAC4) or a truncated form of pUL128 (BAC1), the role of UL128 for infection and motility of monocytes has been investigated. Like TB40E, BAC4 was able to infect and to express IE gene products in monocytes. BAC4-infected monocytes exhibited normal cytoskeleton architecture but a complete inability to migrate in response to chemokines as a consequence of intracellular retention of the cognate chemokine receptors. Similar to TB40F, BAC1 virions entered into monocytes but were retained in cytoplasmatic vesicles and IE gene expression could not be detected. Moreover, the chemokine responsiveness of monocytes inoculated with BAC1 was normal. In order to exclude that second-site mutations were responsible for the observed phenotype, revertant viruses were tested. The re-introduction of the wild-type UL128 sequence in BAC1 resulted in BAC1repaired (BAC1rep) and the introduction of the mutation responsible for the truncation in BAC4 resulted in BAC4mutated (BAC4mut). It has been observed that only BAC4 and BAC1rep, expressing the full-length form of pUL128, were able to infect monocytes and block their migration. Finally, monocytes incubated with the recombinant UL128 protein (rpUL128) showed the same block of migration like BAC4 and BAC1rep infected cells, while monocytes treated with an irrelevant recombinant protein migrated normally. Further more it has been observed that rpUL128 induced monocyte recruitment with the same potency as MCP-1, supporting the hypothesis that pUL128 can act as a chemokine and thus lead to chemokine receptor binding and internalisation

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

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