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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

Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.(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 [46]. 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.
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ppat.1004999.g002: Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.(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 [46]. 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.

Mentions: To determine whether THY-1 is important for HCMV infection, we performed a series of loss-of-function experiments. First, we determined if soluble THY-1 (a.a. 20–130) can block HCMV infection. Wild-type THY-1 is initially synthesized as a 161 amino acid peptide. Upon maturation, the signal peptide (a.a.1-19) is cleaved and the C-terminal a.a. 132–162 is replaced with a GPI anchor. A soluble form of THY-1 (a.a. 20–130) exists in vivo and the recombinant form of THY-1 retains its biological function in binding integrins [39,40]. HCMV or control virus (HSV-2-GFP or adenovirus-GFP) was premixed with soluble THY-1-His protein or a control His protein (soluble varicella-zoster virus gE-His) at room temperature for 10 min, added to HS-578T cells for virus binding on ice for 60 min. Internalization was initiated by raising the temperature to 37°C for 60 min, and then non-absorbed virus was inactivated at low pH, and infectivity was quantified using GFP 3 days later. Compared with the control protein at each dose, soluble THY-1 protein reduced HCMV infectivity in a dose-dependent manner (Figs 2A and S3) in adenocarcinoma cells, and inhibited infection in MRC-5 fibroblasts (Figs 2B and S4 top). In contrast, it did not reduce HSV-2 infectivity (Figs 2C and S4 bottom) or adenovirus infectivity (Figs 2D and S4 Bottom). Soluble THY-1 protein was required during the initial viral entry step to block HCMV infectivity, since addition of the protein after virus binding and internalization did not inhibit infectivity (Fig 2A, last bar). In natural hosts HCMV infection likely occurs at a relatively low m.o.i. A review of studies of virus shedding from saliva of infants, children, and adults, often the source of transmitted virus, showed that the titer of virus in saliva ranged from 103 to 2 x 104 pfu/ml [41]. Therefore we infected the cells with titers ranging from 4 x 104 (HS-578T) to 1 x 105 pfu/ml (MRC-5), which corresponds to a relatively low m.o.i. (0.05 to 1) to try to replicate what may occur during natural infection. Furthermore, we used acid inactivation to limit the infection within the first 60 min to focus on the initial stages of virus infection and the most efficient pathways for viral entry (Fig 2). During the first 60 minutes after infection (m.o.i. 0.05–1 with acid inactivation), about 2–10% of the cells were infected, which corresponds to about 20–35% of the cells if the same infection is allowed to continue for a prolonged time, i.e. without acid inactivation (S5 Fig). Soluble THY-1 protein blocked over 90% of the infection that occurred within the first 60 min (m.o.i. 0.05–1) at a dose of 0.5 μg/ml (Fig 2A). In contrast, with a high m.o.i (4, based on titration in MRC-5 cells) 10-fold more soluble protein was required to block >90% of the infectivity (during entry over 60 min with acid inactivation), and soluble THY-1 blocked infection less efficiently for the virus that enters with slower kinetics (75% reduction in infectivity without acid inactivation, S6 Fig).


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)

Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.(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 [46]. 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.
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ppat.1004999.g002: Soluble THY-1 protein blocks HCMV entry in a dose-dependent manner.(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 [46]. 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.
Mentions: To determine whether THY-1 is important for HCMV infection, we performed a series of loss-of-function experiments. First, we determined if soluble THY-1 (a.a. 20–130) can block HCMV infection. Wild-type THY-1 is initially synthesized as a 161 amino acid peptide. Upon maturation, the signal peptide (a.a.1-19) is cleaved and the C-terminal a.a. 132–162 is replaced with a GPI anchor. A soluble form of THY-1 (a.a. 20–130) exists in vivo and the recombinant form of THY-1 retains its biological function in binding integrins [39,40]. HCMV or control virus (HSV-2-GFP or adenovirus-GFP) was premixed with soluble THY-1-His protein or a control His protein (soluble varicella-zoster virus gE-His) at room temperature for 10 min, added to HS-578T cells for virus binding on ice for 60 min. Internalization was initiated by raising the temperature to 37°C for 60 min, and then non-absorbed virus was inactivated at low pH, and infectivity was quantified using GFP 3 days later. Compared with the control protein at each dose, soluble THY-1 protein reduced HCMV infectivity in a dose-dependent manner (Figs 2A and S3) in adenocarcinoma cells, and inhibited infection in MRC-5 fibroblasts (Figs 2B and S4 top). In contrast, it did not reduce HSV-2 infectivity (Figs 2C and S4 bottom) or adenovirus infectivity (Figs 2D and S4 Bottom). Soluble THY-1 protein was required during the initial viral entry step to block HCMV infectivity, since addition of the protein after virus binding and internalization did not inhibit infectivity (Fig 2A, last bar). In natural hosts HCMV infection likely occurs at a relatively low m.o.i. A review of studies of virus shedding from saliva of infants, children, and adults, often the source of transmitted virus, showed that the titer of virus in saliva ranged from 103 to 2 x 104 pfu/ml [41]. Therefore we infected the cells with titers ranging from 4 x 104 (HS-578T) to 1 x 105 pfu/ml (MRC-5), which corresponds to a relatively low m.o.i. (0.05 to 1) to try to replicate what may occur during natural infection. Furthermore, we used acid inactivation to limit the infection within the first 60 min to focus on the initial stages of virus infection and the most efficient pathways for viral entry (Fig 2). During the first 60 minutes after infection (m.o.i. 0.05–1 with acid inactivation), about 2–10% of the cells were infected, which corresponds to about 20–35% of the cells if the same infection is allowed to continue for a prolonged time, i.e. without acid inactivation (S5 Fig). Soluble THY-1 protein blocked over 90% of the infection that occurred within the first 60 min (m.o.i. 0.05–1) at a dose of 0.5 μg/ml (Fig 2A). In contrast, with a high m.o.i (4, based on titration in MRC-5 cells) 10-fold more soluble protein was required to block >90% of the infectivity (during entry over 60 min with acid inactivation), and soluble THY-1 blocked infection less efficiently for the virus that enters with slower kinetics (75% reduction in infectivity without acid inactivation, S6 Fig).

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