Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.

<p>(A) MRC-5 cells were infected with HCMV AD169 and live cell (surface) staining was performed with goat anti-THY-1 and mouse anti-HCMV gB antibody (row 1), or mouse anti-HCMV gH antibody (row 2). Anti-goat-Alex 594 (red) and anti-mouse Alexa 488 (green) were used to detect THY-1 and HCMV glycoproteins, respectively. Negative controls for non-specific cross-reactivity and background included HCMV-infected MRC-5 cells stained with goat anti-THY-1 and anti-mouse Alexa 488 (row 3a), anti-goat Alexa 594 with mouse anti-HCMV gB (row 3b), or with mouse anti-HCMV gH (row 3c), or anti-goat Alex 594 and anti-mouse Alexa 488 (row 3d). Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). (B) Colocalization of gB with THY-1, but not ZO-1, in HCMV AD169-infected HS-578T adenocarcinoma cells by confocal microscopy. Cell staining was performed with mouse anti-HCMV gB antibody, goat anti-THY-1 antibody, or rabbit anti-ZO-1 antibody followed by anti-mouse Alexa 488 (green), anti-goat Alexa 594 (red), or anti-rabbit Alexa 594. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). 41% of gB colocalized with THY-1, and 44% of THY-1 colocalized with gB; Pearson’s Correlation Coefficient was 0.27; a coefficient of 1.0 indicates 100% colocalization (top row); 3.2% of gB colocalized with ZO-1, and 2.1% of ZO-1 colocalized with gB; Pearson’s coefficient was -0.1 (bottom row).</p

Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.

<p>(A) MRC-5 cells were infected with HCMV AD169 and live cell (surface) staining was performed with goat anti-THY-1 and mouse anti-HCMV gB antibody (row 1), or mouse anti-HCMV gH antibody (row 2). Anti-goat-Alex 594 (red) and anti-mouse Alexa 488 (green) were used to detect THY-1 and HCMV glycoproteins, respectively. Negative controls for non-specific cross-reactivity and background included HCMV-infected MRC-5 cells stained with goat anti-THY-1 and anti-mouse Alexa 488 (row 3a), anti-goat Alexa 594 with mouse anti-HCMV gB (row 3b), or with mouse anti-HCMV gH (row 3c), or anti-goat Alex 594 and anti-mouse Alexa 488 (row 3d). Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). (B) Colocalization of gB with THY-1, but not ZO-1, in HCMV AD169-infected HS-578T adenocarcinoma cells by confocal microscopy. Cell staining was performed with mouse anti-HCMV gB antibody, goat anti-THY-1 antibody, or rabbit anti-ZO-1 antibody followed by anti-mouse Alexa 488 (green), anti-goat Alexa 594 (red), or anti-rabbit Alexa 594. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). 41% of gB colocalized with THY-1, and 44% of THY-1 colocalized with gB; Pearson’s Correlation Coefficient was 0.27; a coefficient of 1.0 indicates 100% colocalization (top row); 3.2% of gB colocalized with ZO-1, and 2.1% of ZO-1 colocalized with gB; Pearson’s coefficient was -0.1 (bottom row).</p

THY-1 Cell Surface Antigen (CD90) Has an Important Role in the Initial Stage of Human Cytomegalovirus Infection

<div><p>Human cytomegalovirus (HCMV) infects about 50% of the US population, is the leading infectious cause of birth defects, and is considered the most important infectious agent in transplant recipients. The virus infects many cell types in vivo and in vitro. While previous studies have identified several cellular proteins that may function at early steps of infection in a cell type dependent manner, the mechanism of virus entry is still poorly understood. Using a computational biology approach, correlating gene expression with virus infectivity in 54 cell lines, we identified THY-1 as a putative host determinant for HCMV infection in these cells. With a series of loss-of-function, gain-of-function and protein-protein interaction analyses, we found that THY-1 mediates HCMV infection at the entry step and is important for infection that occurs at a low m.o.i. THY-1 antibody that bound to the cell surface blocked HCMV during the initial 60 minutes of infection in a dose-dependent manner. Down-regulation of THY-1 with siRNA impaired infectivity occurred during the initial 60 minutes of inoculation. Both THY-1 antibody and siRNA inhibited HCMV-induced activation of the PI3-K/Akt pathway required for entry. Soluble THY-1 protein blocked HCMV infection during, but not after, virus internalization. Expression of exogenous THY-1 enhanced entry in cells expressing low levels of the protein. THY-1 interacted with HCMV gB and gH and may form a complex important for entry. However, since gB and gH have previously been shown to interact, it is uncertain if THY-1 directly binds to both of these proteins. Prior observations that THY-1 (a) interacts with αVβ3 integrin and recruits paxillin (implicated in HCMV entry), (b) regulates leukocyte extravasation (critical for HCMV viremia), and (c) is expressed on many cells targeted for HCMV infection including epithelial and endothelial cells, fibroblast, and CD34+/CD38- stem cells, all support a role for THY-1 as an HCMV entry mediator in a cell type dependent manner. THY-1 may function through a complex setting, that would include viral gB and gH, and other cellular factors, thus links virus entry with signaling in host cells that ultimately leads to virus infection.</p></div

Down-regulation of THY-1 expression blocks HCMV-induced activation of Akt MRC-5 cells were nucleofected with THY-1 specific or control non-targeting siRNAs as described in Fig 1.

<p>48 hrs after transfection, the cells were inoculated with Towne-GFP at 4°C for 60 min for binding and then shifted to 37°C for 15 min to allow synchronized entry. The cells were harvested with lysis buffer (0.1M Tris, 4% SDS and 5% DTT) and proteins were separated on SDS-PAGE gels and immunoblotted sequentially with anti-phosphorylated Akt, anti-actin and anti-THY-1 antibodies. A duplicate membrane was probed with anti-total Akt antibody (A). Densitometry of bands on immunoblots was quantified using ImageJ software (B). A representative experiment is shown from 6 independent experiments performed.</p

Down-regulation of THY-1 expression impairs HCMV entry and exogenous expression of THY-1 enhances entry.

<p>(A) Quantitative RT-PCR of THY-1 expression in SNB-19 (glioblastoma) cells 48 hr after nucleofection of THY-1 specific siRNAs and control non-targeting siRNAs. THY-1 specific siRNAs knocked down THY-1 expression. (B) HCMV infectivity of SNB-19 cells treated with siRNAs for 48 hr and infected with epithelial/endothelial tropic HCMV TB40E-GFP (m.o.i. 0.1). The virus was incubated with cells for 60 min to allow entry before inactivation of virus that had not internalized, and GFP-positive cells were quantified by FACS at day 3 post-infection. Down-regulation of THY-1 by specific siRNAs inhibited HCMV infectivity (P <0.001). 14 independent experiments were performed and a representative result is shown. (C) SF-539 cells were nucleofected with a plasmid expressing THY-1 (pCMV-THY1) or vector control. At 48 hrs post-transfection THY-1 mRNA was quantified by RT-PCR and normalized to GAPDH mRNA amplified in the same reaction. (D) SF-539 cells were infected with Towne-GFP at 48 hr post-transfection for 6 hr and HCMV UL123 and UL55 mRNAs were quantified by RT-PCR and normalized against GAPDH mRNA. Seven independent experiments were carried out and a representative experiment is shown.</p

Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.

<p>(A) HS-578T (adenocarcinoma) cells were infected with Towne-GFP in the presence of increasing amounts of soluble protein. The virus was allowed to enter for 60 min as described in the legend to Fig 2. After culture for 3 days GFP-positivity was quantified by FACS. Equal amounts of THY-1-His and gE-His proteins were used in the assays based on Micro BCA Protein analysis (Pierce, Rockford, IL) and ELISA to determine “His” units [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004999#ppat.1004999.ref046" target="_blank">46</a>]. Seven independent experiments were performed with P value < 0.0001. (B) Towne-GFP HCMV was added to MRC-5 cells at an m.o.i. of 0.1 in the presence of soluble THY-1 (0.25 ug/ml) or control protein sgE (soluble varicella-zoster virus derived gEt-His) or control filtrates obtained during purification of THY-1 protein with an Amicon Ultra centrifugal filter unit (3000 molecular weight cutoff). The virus was allowed to enter the cells for 60 min at 37°C as indicated above followed by low pH citrate buffer wash to inactivate non-internalized virus and remove the soluble proteins. Infectivity was determined by FACS analysis of GFP positive cells at day 3 post-infection. (C) HS-578T cells were infected with HSV-2-GFP (m.o.i. 0.5) in the presence of soluble THY-1 protein (0.5 μg/ml), a control protein (soluble varicella-zoster virus gE, 0.5 μg/ml) or filtrates (derived from THY-1 protein purification in which THY-1 protein was removed by an Amicon Ultra filtration column with a 3000 molecular weight cutoff). Virus infection was performed as described in (A) and then overlaid with 2% human intravenous immune globulin (IVIG) (Talecris Biotherapeutics, Research Triangle Park, NC) and GFP positive cells were determined at 24 hrs post-infection by FACS. (D) HS-578T cells were infected with Towne-GFP or Adenovirus-GFP (0.5–1.0 m.o.i.) in the presence of soluble proteins (0.5 μg/ml), filtrate control, or buffer for 60 min as described in the legend to Fig 2, and the percentage of infected cells was determined by flow cytometry 3 days after infection.</p

Expression of THY-1 positively correlates with HCMV infectivity for both fibroblast and epithelial/endothelial tropic stains of virus in 54 human cell lines.

<p>Cells were infected with both fibroblast tropic HCMV (Towne-GFP) for 2 days and epithelial/endothelial tropic HCMV (TB40E-GFP) for 3 days. We aimed to achieve an infection rate at approximately 10–20% for positive controls without acid inactivation (m.o.i 0.5 for control MRC-5 cells and 0.1–0.5 for ARPE-19 cells). The percentage of infectivity was derived from the mean of three independent infections minus the background florescence contributed by mock-infected cells and normalized against control cell lines (MRC-5 for Towne-GFP, ARPE-19 for TB40E-GFP). THY-1 and PDGFR-α expression levels were determined by multiple microarray platforms (<a href="http://dtp.nci.nih.gov/mtweb" target="_blank">http://dtp.nci.nih.gov/mtweb</a>) and these are baseline levels of RNA in cells not infected with virus. HCMV infectivity positively correlated with THY-1 expression at a level similar to that of PDGFR-α. Cell lines are indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004999#ppat.1004999.s013" target="_blank">S1 Table</a>.</p

THY-1 antibody binds to cell surface THY-1 protein and blocks HCMV entry in a dose-dependent manner.

<p>(A) Total RNA was extracted from different cell lines as described in the Materials and Methods. Quantitative real-time RT-qPCR was performed targeting THY-1 (FAM labeling) and normalized against GAPDH (VIC labeling) which was amplified in the same wells. (B) Cells were incubated with THY-1 specific monoclonal antibody 5E10 or isotype control on ice for 60 min. After washing with cold PBS, the cells were stained with anti-mouse-Alexa-488 conjugate on ice for 30 min, washed, fixed with 2% paraformaldehyde, and analyzed by FACS. (C)–(E) Anti-THY-1 antibody (5E10) or isotype control (25 μg/ml) was added to cells for 60 min on ice for surface binding and then the antibody was washed off. HCMV was added at 0.05 m.o.i. for 60 min at 4°C to allow virus binding, and the temperature was shifted to 37°C for 60 min to allow virus entry. The remaining virus that had not internalized was then inactivated with low pH citrate buffer, and the cells were washed twice with cell culture medium. (C) HS-578T (adenocarcinoma) cells were infected with Towne-GFP virus. RNA was extracted at 6 hr post-infection and HCMV UL123 and UL55 mRNAs were quantified using RT-PCR and normalized against GAPDH mRNA amplified in the same reaction. The experiment was performed 4 times in triplicate, and a representative result is shown here. (D) HS-578T cells were infected with HCMV as described above after incubation with different concentrations of antibody with the cells. After culture for 3 days, GFP-positivity was determined by FACS. The experiment was performed three times in triplicate. (E) MRC-5 cells were pre-incubated with THY-1 antibody 5E10 or IgG control at 25 or 50 ug/ml as described above and infected with TB40E-GFP virus for 60 min before low pH citrate buffer inactivation. Infectivity was assayed for expression of GFP by FACS at 3 days post-infection.</p

HCMV gB and gH, but not ICP8, obtained from infected cell lysates binds to a THY-1 protein column.

<p>(A) Anti-gB antibody detects full length gB (160 kD) and furin cleaved gB (55 kD doublets) in eluates of HCMV-infected cell lysates from THY-1 protein columns with either of two lysis buffers, but not from the VZV gE protein column. The very dark 115 kDa band seen in the THY-1 and gEt bands are background bands likely due to cross-reactivity of the anti-gB or secondary antibody with protein from the His column. (B) Anti-ICP8 antibody detects a 135 kD protein band in lysate from MRC-5 cells infected with HCMV AD169, but not from eluates of lysates applied to THY-1 or VZV gE protein columns. Infected cell lysates were prepared using lysis buffer 1 (PBS with 0.1% NP-40) or lysis buffer 2 (25 mM Tris, 15 mM NaCl and 0.1% NP-40). (C) Anti-gH antibody detects gH (92 kD) in eluates of HCMV-infected cell lysates from THY-1 protein columns with two different lysis buffers. gH was not detected in eluate from a control varicella-zoster gE column.</p

Detection of rhesus LCV-specific CD4 and CD8 T cell responses to gp350 or EBNA-3A and EBNA-3B in rhesus monkeys.

<p>Animals were immunized with soluble gp350, VRP-gp350, a combination of VRP-gp350, VRP-EBNA-3A, and VRP-EBNA-3B, or PBS before challenge with wild-type virus. Pre indicates PBMCs obtained before vaccination; post indicates PBMCs obtained after vaccination.</p