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Identification of novel viral receptors with cell line expressing viral receptor-binding protein.

Mei M, Ye J, Qin A, Wang L, Hu X, Qian K, Shao H - Sci Rep (2015)

Bottom Line: Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease.Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells.Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

View Article: PubMed Central - PubMed

Affiliation: 1] Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, No.12 East Wenhui Road, Yangzhou, Jiangsu. 225009, P. R. China [2] Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, Yangzhou. 225009, P. R. China.

ABSTRACT
The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

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(Qin) chANXA2 binding to ALV-J Env protein (A), Silver Staining of protein precipitation for the membrane proteins of the pcDNA-env_DF1 cells. Lane 1, protein marker; lane 2, precipitated with JE9; lane 3, precipitated with isotype control IgG. (B), The fusion protein SUJ-rIgGFc was analyzed by western blotting with JE9. Lane1, protein marker; lane2, lysate of MDCK cells infected with rAd-SUJ-rIgGFc; lane3, lysate of MDCK cells infected with wild type rAd; (C), Silver Staining of protein precipitation for the membrane proteins of DF1 cells. Lane 1, protein marker; lane 2, precipitated with SUJ-rIgGFc; lane 3, precipitated with rabbit IgG. (D), Western blot assay for the co-immunoprecipitation. Lane 1, 293T cell transfected with pcDNA3.1-EnvJ were analyzed with JE9; lane 2, 293T cell transfected with chANXA2 were analysed with anti-chANXA2 (C-16); lane 3, 293T cell lysates transfected with chANXA2 immunoprecipitated with JE9 and analyzed with anti-chANXA2 (C-16); lane 4, 293T cell co-transfected with pcDNA3.1-EnvJ and chANXA2 were immunoprecipitated with JE9, and analyzed with JE9 and anti-chANXA2 (C-16).
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f1: (Qin) chANXA2 binding to ALV-J Env protein (A), Silver Staining of protein precipitation for the membrane proteins of the pcDNA-env_DF1 cells. Lane 1, protein marker; lane 2, precipitated with JE9; lane 3, precipitated with isotype control IgG. (B), The fusion protein SUJ-rIgGFc was analyzed by western blotting with JE9. Lane1, protein marker; lane2, lysate of MDCK cells infected with rAd-SUJ-rIgGFc; lane3, lysate of MDCK cells infected with wild type rAd; (C), Silver Staining of protein precipitation for the membrane proteins of DF1 cells. Lane 1, protein marker; lane 2, precipitated with SUJ-rIgGFc; lane 3, precipitated with rabbit IgG. (D), Western blot assay for the co-immunoprecipitation. Lane 1, 293T cell transfected with pcDNA3.1-EnvJ were analyzed with JE9; lane 2, 293T cell transfected with chANXA2 were analysed with anti-chANXA2 (C-16); lane 3, 293T cell lysates transfected with chANXA2 immunoprecipitated with JE9 and analyzed with anti-chANXA2 (C-16); lane 4, 293T cell co-transfected with pcDNA3.1-EnvJ and chANXA2 were immunoprecipitated with JE9, and analyzed with JE9 and anti-chANXA2 (C-16).

Mentions: The pcDNA-env_DF1 cell line expressing ALV-J Env protein was previously constructed and shown to be resistant to ALV-J infection18. To use this cell line to isolate novel functional receptors for ALV-J, we first extracted the membrane proteins from the pcDNA-env_DF1 cells and then performed immunoprecipitation with the single monoclonal antibody (mAb) JE-9, which is specific to ALV-J Env19. Silver staining for SDS-PAGE of the immunoprecipitation revealed several different bands in the lysate that was immunoprecipitated with ALV-J-specific mAb JE-9 and not with the control antibody (Fig. 1A). Mass spectrometry further revealed that one of these bands was chicken Annexin A2 (chANXA2), a member of the annexin family20.


Identification of novel viral receptors with cell line expressing viral receptor-binding protein.

Mei M, Ye J, Qin A, Wang L, Hu X, Qian K, Shao H - Sci Rep (2015)

(Qin) chANXA2 binding to ALV-J Env protein (A), Silver Staining of protein precipitation for the membrane proteins of the pcDNA-env_DF1 cells. Lane 1, protein marker; lane 2, precipitated with JE9; lane 3, precipitated with isotype control IgG. (B), The fusion protein SUJ-rIgGFc was analyzed by western blotting with JE9. Lane1, protein marker; lane2, lysate of MDCK cells infected with rAd-SUJ-rIgGFc; lane3, lysate of MDCK cells infected with wild type rAd; (C), Silver Staining of protein precipitation for the membrane proteins of DF1 cells. Lane 1, protein marker; lane 2, precipitated with SUJ-rIgGFc; lane 3, precipitated with rabbit IgG. (D), Western blot assay for the co-immunoprecipitation. Lane 1, 293T cell transfected with pcDNA3.1-EnvJ were analyzed with JE9; lane 2, 293T cell transfected with chANXA2 were analysed with anti-chANXA2 (C-16); lane 3, 293T cell lysates transfected with chANXA2 immunoprecipitated with JE9 and analyzed with anti-chANXA2 (C-16); lane 4, 293T cell co-transfected with pcDNA3.1-EnvJ and chANXA2 were immunoprecipitated with JE9, and analyzed with JE9 and anti-chANXA2 (C-16).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300512&req=5

f1: (Qin) chANXA2 binding to ALV-J Env protein (A), Silver Staining of protein precipitation for the membrane proteins of the pcDNA-env_DF1 cells. Lane 1, protein marker; lane 2, precipitated with JE9; lane 3, precipitated with isotype control IgG. (B), The fusion protein SUJ-rIgGFc was analyzed by western blotting with JE9. Lane1, protein marker; lane2, lysate of MDCK cells infected with rAd-SUJ-rIgGFc; lane3, lysate of MDCK cells infected with wild type rAd; (C), Silver Staining of protein precipitation for the membrane proteins of DF1 cells. Lane 1, protein marker; lane 2, precipitated with SUJ-rIgGFc; lane 3, precipitated with rabbit IgG. (D), Western blot assay for the co-immunoprecipitation. Lane 1, 293T cell transfected with pcDNA3.1-EnvJ were analyzed with JE9; lane 2, 293T cell transfected with chANXA2 were analysed with anti-chANXA2 (C-16); lane 3, 293T cell lysates transfected with chANXA2 immunoprecipitated with JE9 and analyzed with anti-chANXA2 (C-16); lane 4, 293T cell co-transfected with pcDNA3.1-EnvJ and chANXA2 were immunoprecipitated with JE9, and analyzed with JE9 and anti-chANXA2 (C-16).
Mentions: The pcDNA-env_DF1 cell line expressing ALV-J Env protein was previously constructed and shown to be resistant to ALV-J infection18. To use this cell line to isolate novel functional receptors for ALV-J, we first extracted the membrane proteins from the pcDNA-env_DF1 cells and then performed immunoprecipitation with the single monoclonal antibody (mAb) JE-9, which is specific to ALV-J Env19. Silver staining for SDS-PAGE of the immunoprecipitation revealed several different bands in the lysate that was immunoprecipitated with ALV-J-specific mAb JE-9 and not with the control antibody (Fig. 1A). Mass spectrometry further revealed that one of these bands was chicken Annexin A2 (chANXA2), a member of the annexin family20.

Bottom Line: Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease.Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells.Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

View Article: PubMed Central - PubMed

Affiliation: 1] Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, No.12 East Wenhui Road, Yangzhou, Jiangsu. 225009, P. R. China [2] Key Laboratory of Jiangsu Preventive Veterinary Medicine, Yangzhou University, Yangzhou. 225009, P. R. China.

ABSTRACT
The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

Show MeSH
Related in: MedlinePlus