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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 impairs HCMV entry and exogenous expression of THY-1 enhances entry.(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.
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ppat.1004999.g004: Down-regulation of THY-1 expression impairs HCMV entry and exogenous expression of THY-1 enhances entry.(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.

Mentions: To confirm the loss-of-function findings observed with THY-1 specific antibody, we used THY-1 specific siRNAs to knockdown THY-1 expression in permissive cells, and analyzed the effect on HCMV infection. Nucleofection of cells with THY-1 specific siRNAs reduced THY-1 expression by over 90% compared with control siRNAs at the time of infection both at the mRNA (Fig 4A) and protein level (S10A Fig and see section on THY-1 and Akt activation below). HCMV infectivity was reduced by 30–50% at 3 days post-infection based on FACS analysis for GFP (Fig 4B) (P value <0.0001, 12 independent experiments). However, THY-1 siRNAs knocked down cell surface THY-1 protein (S10B Fig) less effectively than total THY-1 protein (S10A Fig). This might be due to increased stability of surface THY-1 protein when it is anchored into lipid rafts, and could contribute to the lower level of inhibition of HCMV infection with THY-1 siRNAs than with antibody or soluble protein (see above). The impairment of HCMV infectivity following knockdown of THY-1 was observed in glioblastoma (SNB-19), adenocarcinoma (HS-578T) and MRC-5 cells infected with either epithelial/endothelial or fibroblast tropic HCMV. Since the infection protocol allowed only 60 min for virus entry before virus was inactivated by low pH, the reduction of infectivity occurred during initiation of HCMV infection.


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 impairs HCMV entry and exogenous expression of THY-1 enhances entry.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492587&req=5

ppat.1004999.g004: Down-regulation of THY-1 expression impairs HCMV entry and exogenous expression of THY-1 enhances entry.(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.
Mentions: To confirm the loss-of-function findings observed with THY-1 specific antibody, we used THY-1 specific siRNAs to knockdown THY-1 expression in permissive cells, and analyzed the effect on HCMV infection. Nucleofection of cells with THY-1 specific siRNAs reduced THY-1 expression by over 90% compared with control siRNAs at the time of infection both at the mRNA (Fig 4A) and protein level (S10A Fig and see section on THY-1 and Akt activation below). HCMV infectivity was reduced by 30–50% at 3 days post-infection based on FACS analysis for GFP (Fig 4B) (P value <0.0001, 12 independent experiments). However, THY-1 siRNAs knocked down cell surface THY-1 protein (S10B Fig) less effectively than total THY-1 protein (S10A Fig). This might be due to increased stability of surface THY-1 protein when it is anchored into lipid rafts, and could contribute to the lower level of inhibition of HCMV infection with THY-1 siRNAs than with antibody or soluble protein (see above). The impairment of HCMV infectivity following knockdown of THY-1 was observed in glioblastoma (SNB-19), adenocarcinoma (HS-578T) and MRC-5 cells infected with either epithelial/endothelial or fibroblast tropic HCMV. Since the infection protocol allowed only 60 min for virus entry before virus was inactivated by low pH, the reduction of infectivity occurred during initiation of HCMV infection.

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