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

Li Q, Wilkie AR, Weller M, Liu X, Cohen JI - PLoS Pathog. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

THY-1 antibody binds to cell surface THY-1 protein and blocks HCMV entry in a dose-dependent manner.(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.
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ppat.1004999.g003: THY-1 antibody binds to cell surface THY-1 protein and blocks HCMV entry in a dose-dependent manner.(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.

Mentions: Next, we examined whether specific antibody 5E10 binds to cell surface THY-1 protein. NCI-60 cell lines SNB-19 (glioblastoma) and HS-578T (adenocarcinoma), as well as primary human diploid (MRC-5) fibroblasts all express THY-1 mRNA [34] (Fig 3A), and THY-1 protein was detected on the surface of these cells (Figs 3B and S1B). Both HS-578T and SNB-19 cells support productive HCMV infection and produce progeny virus (S2 Fig), although HCMV cell-to-cell spread in SNB-19 cells is limited, especially with TB40E-GFP HCMV. To ascertain whether THY-1 specific antibody blocks HCMV infection, THY-1 or isotype control antibody was allowed to bind to the surface of HS-578T cells on ice for 60 min, the antibody mixture was removed from the cells, and HCMV was added on ice for 60 min to synchronize virus binding. To focus on the early steps of virus entry, the temperature was raised to 37°C for 60 min to allow virus entry, followed by low pH treatment to inactivate any virions that still remained on the cell surface or in the medium. After washing, the cells were then cultured for 6 hr before RNA extraction to quantify combined HCMV UL123 (encodes IE1) and UL55 (encodes gB) RNA expression by RT-qPCR [42] or for 3 days to measure infectivity by FACS for GFP. Although UL55 is a late gene, UL55 transcripts start to appear at 4 hrs post-infection, and expression is not strictly dependent on new viral DNA synthesis [43,44]. In 4 independent experiments, quantitative RT-PCR showed that THY-1 specific antibody blocked expression of HCMV UL123 and UL55 genes, compared with isotype control antibody (Fig 3C, P = 0.0002 for 4 independent experiments). Similar blocking result with THY-1 antibody was also seen when infectivity was assayed at 3 days post-infection by virus-encoded GFP (Fig 3D, P = 0.0004, 3 independent experiments). THY-1 specific antibody, but not isotype control, blocked HCMV infectivity in a dose-dependent manner (Figs 3D and S7). THY-1 antibody also blocked HCMV infection in primary MRC-5 cells (Fig 3E).


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

Li Q, Wilkie AR, Weller M, Liu X, Cohen JI - PLoS Pathog. (2015)

THY-1 antibody binds to cell surface THY-1 protein and blocks HCMV entry in a dose-dependent manner.(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.
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getmorefigures.php?uid=PMC4492587&req=5

ppat.1004999.g003: THY-1 antibody binds to cell surface THY-1 protein and blocks HCMV entry in a dose-dependent manner.(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.
Mentions: Next, we examined whether specific antibody 5E10 binds to cell surface THY-1 protein. NCI-60 cell lines SNB-19 (glioblastoma) and HS-578T (adenocarcinoma), as well as primary human diploid (MRC-5) fibroblasts all express THY-1 mRNA [34] (Fig 3A), and THY-1 protein was detected on the surface of these cells (Figs 3B and S1B). Both HS-578T and SNB-19 cells support productive HCMV infection and produce progeny virus (S2 Fig), although HCMV cell-to-cell spread in SNB-19 cells is limited, especially with TB40E-GFP HCMV. To ascertain whether THY-1 specific antibody blocks HCMV infection, THY-1 or isotype control antibody was allowed to bind to the surface of HS-578T cells on ice for 60 min, the antibody mixture was removed from the cells, and HCMV was added on ice for 60 min to synchronize virus binding. To focus on the early steps of virus entry, the temperature was raised to 37°C for 60 min to allow virus entry, followed by low pH treatment to inactivate any virions that still remained on the cell surface or in the medium. After washing, the cells were then cultured for 6 hr before RNA extraction to quantify combined HCMV UL123 (encodes IE1) and UL55 (encodes gB) RNA expression by RT-qPCR [42] or for 3 days to measure infectivity by FACS for GFP. Although UL55 is a late gene, UL55 transcripts start to appear at 4 hrs post-infection, and expression is not strictly dependent on new viral DNA synthesis [43,44]. In 4 independent experiments, quantitative RT-PCR showed that THY-1 specific antibody blocked expression of HCMV UL123 and UL55 genes, compared with isotype control antibody (Fig 3C, P = 0.0002 for 4 independent experiments). Similar blocking result with THY-1 antibody was also seen when infectivity was assayed at 3 days post-infection by virus-encoded GFP (Fig 3D, P = 0.0004, 3 independent experiments). THY-1 specific antibody, but not isotype control, blocked HCMV infectivity in a dose-dependent manner (Figs 3D and S7). THY-1 antibody also blocked HCMV infection in primary MRC-5 cells (Fig 3E).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Virology Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus