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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

HCMV gB and gH, but not ICP8, obtained from infected cell lysates binds to a THY-1 protein column.(A) Anti-gB antibody detects full length gB (160 kD) and furin cleaved gB (55 kD doublets) in eluates of HCMV-infected cell lysates from THY-1 protein columns with either of two lysis buffers, but not from the VZV gE protein column. The very dark 115 kDa band seen in the THY-1 and gEt bands are background bands likely due to cross-reactivity of the anti-gB or secondary antibody with protein from the His column. (B) Anti-ICP8 antibody detects a 135 kD protein band in lysate from MRC-5 cells infected with HCMV AD169, but not from eluates of lysates applied to THY-1 or VZV gE protein columns. Infected cell lysates were prepared using lysis buffer 1 (PBS with 0.1% NP-40) or lysis buffer 2 (25 mM Tris, 15 mM NaCl and 0.1% NP-40). (C) Anti-gH antibody detects gH (92 kD) in eluates of HCMV-infected cell lysates from THY-1 protein columns with two different lysis buffers. gH was not detected in eluate from a control varicella-zoster gE column.
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ppat.1004999.g005: HCMV gB and gH, but not ICP8, obtained from infected cell lysates binds to a THY-1 protein column.(A) Anti-gB antibody detects full length gB (160 kD) and furin cleaved gB (55 kD doublets) in eluates of HCMV-infected cell lysates from THY-1 protein columns with either of two lysis buffers, but not from the VZV gE protein column. The very dark 115 kDa band seen in the THY-1 and gEt bands are background bands likely due to cross-reactivity of the anti-gB or secondary antibody with protein from the His column. (B) Anti-ICP8 antibody detects a 135 kD protein band in lysate from MRC-5 cells infected with HCMV AD169, but not from eluates of lysates applied to THY-1 or VZV gE protein columns. Infected cell lysates were prepared using lysis buffer 1 (PBS with 0.1% NP-40) or lysis buffer 2 (25 mM Tris, 15 mM NaCl and 0.1% NP-40). (C) Anti-gH antibody detects gH (92 kD) in eluates of HCMV-infected cell lysates from THY-1 protein columns with two different lysis buffers. gH was not detected in eluate from a control varicella-zoster gE column.

Mentions: Since co-immunoprecipitation followed by Western blotting was inefficient for detecting proteins that interact with gB in infected cells, we constructed protein columns by binding THY-1-His protein, or control VZV gE-His protein to Talon beads, added lysates from HCMV-infected cells to the columns, eluted proteins bound to the columns, and immunoblotted the proteins with antibody to HCMV ICP8 or gB. Two different cell lysis buffers were used, PBS with 0.1% NP-40 [30] and 25 mM Tris, 15 mM NaCl and 0.1% NP-40 [46]. HCMV gB was detected in the infected cell lysate and in eluates from THY-1 protein columns, but not the control VZV gE protein column (Fig 5A). Interestingly, THY-1 complexed with full length gB (160 kD), as well as its proteolytic cleavage products of 55 kD [47]. Purified THY-1 protein pulled down more 55 kD gB than full length gB. A previous study has shown the cleaved form of gB was more abundant than full length gB in infected cell lysate and in purified virions [48]. In contrast, the 135 kD HCMV ICP8 was detected in infected cell lysate, but not in eluates from THY-1 or control VZV gE protein columns (Fig 5B). Similarly, gH was co-precipitated from infected cell lysate by purified THY-1 protein (Fig 5C). These results suggest that THY-1 may form a complex with HCMV gB and gH in infected cells. Since gB and gH have been shown to form a complex, it is possible that THY-1 interacts directly with gB and that the interaction of THY-1 with gH is indirect and solely due to gH interacting with gB. Alternatively, THY-1 has been shown to bind to integrins, and gB and gH from several herpesviruses interact with integrins; thus, the interaction between THY-1 and gB and gH may be indirect and mediated through integrins.


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)

HCMV gB and gH, but not ICP8, obtained from infected cell lysates binds to a THY-1 protein column.(A) Anti-gB antibody detects full length gB (160 kD) and furin cleaved gB (55 kD doublets) in eluates of HCMV-infected cell lysates from THY-1 protein columns with either of two lysis buffers, but not from the VZV gE protein column. The very dark 115 kDa band seen in the THY-1 and gEt bands are background bands likely due to cross-reactivity of the anti-gB or secondary antibody with protein from the His column. (B) Anti-ICP8 antibody detects a 135 kD protein band in lysate from MRC-5 cells infected with HCMV AD169, but not from eluates of lysates applied to THY-1 or VZV gE protein columns. Infected cell lysates were prepared using lysis buffer 1 (PBS with 0.1% NP-40) or lysis buffer 2 (25 mM Tris, 15 mM NaCl and 0.1% NP-40). (C) Anti-gH antibody detects gH (92 kD) in eluates of HCMV-infected cell lysates from THY-1 protein columns with two different lysis buffers. gH was not detected in eluate from a control varicella-zoster gE column.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004999.g005: HCMV gB and gH, but not ICP8, obtained from infected cell lysates binds to a THY-1 protein column.(A) Anti-gB antibody detects full length gB (160 kD) and furin cleaved gB (55 kD doublets) in eluates of HCMV-infected cell lysates from THY-1 protein columns with either of two lysis buffers, but not from the VZV gE protein column. The very dark 115 kDa band seen in the THY-1 and gEt bands are background bands likely due to cross-reactivity of the anti-gB or secondary antibody with protein from the His column. (B) Anti-ICP8 antibody detects a 135 kD protein band in lysate from MRC-5 cells infected with HCMV AD169, but not from eluates of lysates applied to THY-1 or VZV gE protein columns. Infected cell lysates were prepared using lysis buffer 1 (PBS with 0.1% NP-40) or lysis buffer 2 (25 mM Tris, 15 mM NaCl and 0.1% NP-40). (C) Anti-gH antibody detects gH (92 kD) in eluates of HCMV-infected cell lysates from THY-1 protein columns with two different lysis buffers. gH was not detected in eluate from a control varicella-zoster gE column.
Mentions: Since co-immunoprecipitation followed by Western blotting was inefficient for detecting proteins that interact with gB in infected cells, we constructed protein columns by binding THY-1-His protein, or control VZV gE-His protein to Talon beads, added lysates from HCMV-infected cells to the columns, eluted proteins bound to the columns, and immunoblotted the proteins with antibody to HCMV ICP8 or gB. Two different cell lysis buffers were used, PBS with 0.1% NP-40 [30] and 25 mM Tris, 15 mM NaCl and 0.1% NP-40 [46]. HCMV gB was detected in the infected cell lysate and in eluates from THY-1 protein columns, but not the control VZV gE protein column (Fig 5A). Interestingly, THY-1 complexed with full length gB (160 kD), as well as its proteolytic cleavage products of 55 kD [47]. Purified THY-1 protein pulled down more 55 kD gB than full length gB. A previous study has shown the cleaved form of gB was more abundant than full length gB in infected cell lysate and in purified virions [48]. In contrast, the 135 kD HCMV ICP8 was detected in infected cell lysate, but not in eluates from THY-1 or control VZV gE protein columns (Fig 5B). Similarly, gH was co-precipitated from infected cell lysate by purified THY-1 protein (Fig 5C). These results suggest that THY-1 may form a complex with HCMV gB and gH in infected cells. Since gB and gH have been shown to form a complex, it is possible that THY-1 interacts directly with gB and that the interaction of THY-1 with gH is indirect and solely due to gH interacting with gB. Alternatively, THY-1 has been shown to bind to integrins, and gB and gH from several herpesviruses interact with integrins; thus, the interaction between THY-1 and gB and gH may be indirect and mediated through integrins.

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