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

Impact of sequence variation in the ul128 locus on production of human cytomegalovirus in fibroblast and epithelial cells

The human cytomegalovirus (HCMV) virion envelope contains a complex consisting of glycoproteins gH and gL plus proteins encoded by the UL128 locus (UL128L): pUL128, pUL130, and pUL131A. UL128L is necessary for efficient infection of myeloid, epithelial, and endothelial cells but limits replication in fibroblasts. Consequently, disrupting mutations in UL128L are rapidly selected when clinical isolates are cultured in fibroblasts. In contrast, bacterial artificial chromosome (BAC)-cloned strains TB40-BAC4, FIX, and TR do not contain overt disruptions in UL128L, yet no virus reconstituted from them has been reported to acquire mutations in UL128L in vitro. We performed BAC mutagenesis and reconstitution experiments to test the hypothesis that these strains contain subtle mutations in UL128L that were acquired during passage prior to BAC cloning. Compared to strain Merlin containing wild-type UL128L, all three strains produced higher yields of cell-free virus. Moreover, TB40-BAC4 and FIX spread cell to cell more rapidly than wild-type Merlin in fibroblasts but more slowly in epithelial cells. The differential growth properties of TB40-BAC4 and FIX (but not TR) were mapped to single-nucleotide substitutions in UL128L. The substitution in TB40-BAC4 reduced the splicing efficiency of UL128, and that in FIX resulted in an amino acid substitution in UL130. Introduction of these substitutions into Merlin dramatically increased yields of cell-free virus and increased cell-to-cell spread in fibroblasts but reduced the abundance of pUL128 in the virion and the efficiency of epithelial cell infection. These substitutions appear to represent mutations in UL128L that permit virus to be propagated in fibroblasts while retaining epithelial cell tropism

Human cytomegalovirus infection of langerhans-type dendritic cells does not require the presence of the gH/gL/UL128-131A complex and is blocked after nuclear deposition of viral genomes in immature cells

Human cytomegalovirus (CMV) enters its host via the oral and genital mucosae. Langerhans-type dendritic cells (LC) are the most abundant innate immune cells at these sites, where they constitute a first line of defense against a variety of pathogens. We previously showed that immature LC (iLC) are remarkably resistant to CMV infection, while mature LC (mLC) are more permissive, particularly when exposed to clinical-strain-like strains of CMV, which display a pentameric complex consisting of the viral glycoproteins gH, gL, UL128, UL130, and UL131A on their envelope. This complex was recently shown to be required for the infection of immature monocyte-derived dendritic cells. We thus sought to establish if the presence of this complex is also necessary for virion penetration of LC and if defects in entry might be the source of iLC resistance to CMV. Here we report that the efficiency of LC infection is reduced, but not completely abolished, in the absence of the pentameric complex. While virion penetration and nuclear deposition of viral genomes are not impaired in iLC, the transcription of the viral immediate early genes UL122 and UL123 and of the delayed early gene UL50 is substantially lower than that in mLC. Together, these data show that the UL128, UL130, and UL131A proteins are dispensable for CMV entry into LC and that progression of the viral cycle in iLC is restricted at the step of viral gene expression

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

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

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

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

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