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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

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 1.48 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.
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ppat.1004999.g008: 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 1.48 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.

Mentions: Previous studies have shown THY-1 modulates the phosphatidylinositol 3-kinase (PI3K) signaling pathway [50]. Activation of the PI3K pathway is required for HCMV infection at the entry step [14,20,22]. Therefore, we analyzed the effect of THY-1 on the ability of HCMV to phosphorylate Akt, a downstream molecule in the PI3K pathway. Knock-down of THY-1 expression with specific siRNAs blocked HCMV-induced phosphorylation of Akt at 15 min post-infection and reduced HCMV infectivity within the first 60 min of infection (Figs 8A and 8B and S12) compared with control siRNAs (p = 0.01, 6 independent experiments). These data suggest that HCMV engagement of THY-1 during the initial 15 min of infection contributes to HCMV signaling through the PI3K/Akt pathway.


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)

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 1.48 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492587&req=5

ppat.1004999.g008: 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 1.48 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.
Mentions: Previous studies have shown THY-1 modulates the phosphatidylinositol 3-kinase (PI3K) signaling pathway [50]. Activation of the PI3K pathway is required for HCMV infection at the entry step [14,20,22]. Therefore, we analyzed the effect of THY-1 on the ability of HCMV to phosphorylate Akt, a downstream molecule in the PI3K pathway. Knock-down of THY-1 expression with specific siRNAs blocked HCMV-induced phosphorylation of Akt at 15 min post-infection and reduced HCMV infectivity within the first 60 min of infection (Figs 8A and 8B and S12) compared with control siRNAs (p = 0.01, 6 independent experiments). These data suggest that HCMV engagement of THY-1 during the initial 15 min of infection contributes to HCMV signaling through the PI3K/Akt pathway.

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