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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

Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.(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).
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ppat.1004999.g006: Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.(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).

Mentions: To further study the possibility of an interaction between THY-1 and the HCMV gB and gH glycoproteins [10], MRC-5 cells were infected with HCMV AD169 (which does not express GFP) and live cell staining was performed with goat anti-THY-1 antibody and mouse monoclonal anti-gB, anti-gH, or isotype control antibody followed by anti-goat and anti-mouse fluorescent antibodies and confocal microscopy. THY-1 colocalized with gB (Pearson Correlation Coefficient 0.88 where 1.0 is 100% colocalization [49] (Fig 6A, row 1) and gH (Pearson Correlation Coefficient 0.84, Fig 6A row 2). Incubation of MRC-5 cells with secondary antibody alone did not give background staining, goat anti-THY-1 did not cross react with secondary anti-mouse fluorescent antibody, and mouse anti-glycoprotein antibodies did not cross react with secondary anti-goat fluorescent antibody (Fig 6A, row 3). In HCMV- infected adenocarcinoma HS-578T cells, gB also colocalized with THY-1 (Figs 6B and S11). As a control, gB did not colocalize with cell surface protein ZO-1 (Fig 6B). Interestingly, confocal microscopy with 3-D reconstruction of the cell surface showed that gB appeared to bind predominantly on top of THY-1 molecules on the plasma membrane (Fig 7). Although gB is conserved among human herpesviruses, HCMV gB (AD169 strain) and VZV gB (Dumas strain) share only 20% amino acid identity and 31% similarity. As an additional control, we co-transfected THY-1 with either HCMV gB or VZV gB, and performed confocal microscopy. HCMV gB colocalized with THY-1 at levels similar to that in infected cells, but VZV gB did not colocalize with THY-1 (S11 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)

Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.(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).
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ppat.1004999.g006: Colocalization of THY-1 with HCMV gB and gH from virus-infected cells by confocal microscopy.(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).
Mentions: To further study the possibility of an interaction between THY-1 and the HCMV gB and gH glycoproteins [10], MRC-5 cells were infected with HCMV AD169 (which does not express GFP) and live cell staining was performed with goat anti-THY-1 antibody and mouse monoclonal anti-gB, anti-gH, or isotype control antibody followed by anti-goat and anti-mouse fluorescent antibodies and confocal microscopy. THY-1 colocalized with gB (Pearson Correlation Coefficient 0.88 where 1.0 is 100% colocalization [49] (Fig 6A, row 1) and gH (Pearson Correlation Coefficient 0.84, Fig 6A row 2). Incubation of MRC-5 cells with secondary antibody alone did not give background staining, goat anti-THY-1 did not cross react with secondary anti-mouse fluorescent antibody, and mouse anti-glycoprotein antibodies did not cross react with secondary anti-goat fluorescent antibody (Fig 6A, row 3). In HCMV- infected adenocarcinoma HS-578T cells, gB also colocalized with THY-1 (Figs 6B and S11). As a control, gB did not colocalize with cell surface protein ZO-1 (Fig 6B). Interestingly, confocal microscopy with 3-D reconstruction of the cell surface showed that gB appeared to bind predominantly on top of THY-1 molecules on the plasma membrane (Fig 7). Although gB is conserved among human herpesviruses, HCMV gB (AD169 strain) and VZV gB (Dumas strain) share only 20% amino acid identity and 31% similarity. As an additional control, we co-transfected THY-1 with either HCMV gB or VZV gB, and performed confocal microscopy. HCMV gB colocalized with THY-1 at levels similar to that in infected cells, but VZV gB did not colocalize with THY-1 (S11 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