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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) Inhibition of ALV-J infection by antibodies to ANXA2.The DF1 cells that had been pre-treated with antibodies against ANXA2 were infected with ALV-J or ALV-A, and the replications of ALV-J and ALV-A in the treated cells were analysed. (A), IFA analysis using JE9; (B), TCID50 analysis for viral titres; (C), western blot analysis for the expression of Env from ALV-J in the DF1 cells that were treated with antibodies. Lane1, mock; lane 2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (5 μg/ml); lane 4, anti-ANXA2 (25 μg/ml); lane 5, anti-ANXA2 (50 μg/ml); (D), western blot analysis for the expression of p27 of ALV-A in the DF1 cells treated with antibodies. Lane 1, mock; lane2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (50 μg/ml).
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f2: (Qin) Inhibition of ALV-J infection by antibodies to ANXA2.The DF1 cells that had been pre-treated with antibodies against ANXA2 were infected with ALV-J or ALV-A, and the replications of ALV-J and ALV-A in the treated cells were analysed. (A), IFA analysis using JE9; (B), TCID50 analysis for viral titres; (C), western blot analysis for the expression of Env from ALV-J in the DF1 cells that were treated with antibodies. Lane1, mock; lane 2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (5 μg/ml); lane 4, anti-ANXA2 (25 μg/ml); lane 5, anti-ANXA2 (50 μg/ml); (D), western blot analysis for the expression of p27 of ALV-A in the DF1 cells treated with antibodies. Lane 1, mock; lane2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (50 μg/ml).

Mentions: To test whether chANXA2 serves as a functional receptor for ALV-J infection, we used antibodies against chANXA2 to perform blocking assays to evaluate the effects of chANXA2 on ALV-J infection in DF1 cells. Our results revealed that the viral infection/replication of ALV-J was significantly inhibited in groups that had been treated with anti-ANXA2. As shown in Fig. 2A, there was little visible immunofluorescence in the cells that were treated with 50 or 25 μg/ml of the antibody against chANXA2 in the IFA. Moreover, only a few positive cells were found among the cells that were treated with 5 μg/ml of antibody against ANXA2. In contrast, many positive cells were found among the cells that were treated with the control IgG and among the untreated cells. Consistent with the IFA results, the viral titres of the cells that were treated with 50 and 25 μg/ml of antibody against chANXA2 were approximately 50-fold and 10-fold less, respectively, than those of the cells that were treated with the control IgG (Fig. 2B). The inhibitory effect on ALV-J infection/replication conferred by the antibody against ANXA2 was also confirmed by western blot (Fig. 2C). As a viral control, we also performed a blocking assay for ALV-A infection in the DF1 cells. As described in Fig. 2D, the p27 expression levels of ALV-A in the groups that were treated with the antibody against chANXA2 were similar to those of the mock group, which indicates that the antibody against ANXA2 could not inhibit ALV-A infection/replication in DF1 cells. These data clearly demonstrate that blocking chANXA2 with a specific antibody can effectively and specially inhibit the infection/replication of ALV-J.


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) Inhibition of ALV-J infection by antibodies to ANXA2.The DF1 cells that had been pre-treated with antibodies against ANXA2 were infected with ALV-J or ALV-A, and the replications of ALV-J and ALV-A in the treated cells were analysed. (A), IFA analysis using JE9; (B), TCID50 analysis for viral titres; (C), western blot analysis for the expression of Env from ALV-J in the DF1 cells that were treated with antibodies. Lane1, mock; lane 2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (5 μg/ml); lane 4, anti-ANXA2 (25 μg/ml); lane 5, anti-ANXA2 (50 μg/ml); (D), western blot analysis for the expression of p27 of ALV-A in the DF1 cells treated with antibodies. Lane 1, mock; lane2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (50 μg/ml).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: (Qin) Inhibition of ALV-J infection by antibodies to ANXA2.The DF1 cells that had been pre-treated with antibodies against ANXA2 were infected with ALV-J or ALV-A, and the replications of ALV-J and ALV-A in the treated cells were analysed. (A), IFA analysis using JE9; (B), TCID50 analysis for viral titres; (C), western blot analysis for the expression of Env from ALV-J in the DF1 cells that were treated with antibodies. Lane1, mock; lane 2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (5 μg/ml); lane 4, anti-ANXA2 (25 μg/ml); lane 5, anti-ANXA2 (50 μg/ml); (D), western blot analysis for the expression of p27 of ALV-A in the DF1 cells treated with antibodies. Lane 1, mock; lane2, goat IgG (50 μg/ml); lane 3, anti-ANXA2 (50 μg/ml).
Mentions: To test whether chANXA2 serves as a functional receptor for ALV-J infection, we used antibodies against chANXA2 to perform blocking assays to evaluate the effects of chANXA2 on ALV-J infection in DF1 cells. Our results revealed that the viral infection/replication of ALV-J was significantly inhibited in groups that had been treated with anti-ANXA2. As shown in Fig. 2A, there was little visible immunofluorescence in the cells that were treated with 50 or 25 μg/ml of the antibody against chANXA2 in the IFA. Moreover, only a few positive cells were found among the cells that were treated with 5 μg/ml of antibody against ANXA2. In contrast, many positive cells were found among the cells that were treated with the control IgG and among the untreated cells. Consistent with the IFA results, the viral titres of the cells that were treated with 50 and 25 μg/ml of antibody against chANXA2 were approximately 50-fold and 10-fold less, respectively, than those of the cells that were treated with the control IgG (Fig. 2B). The inhibitory effect on ALV-J infection/replication conferred by the antibody against ANXA2 was also confirmed by western blot (Fig. 2C). As a viral control, we also performed a blocking assay for ALV-A infection in the DF1 cells. As described in Fig. 2D, the p27 expression levels of ALV-A in the groups that were treated with the antibody against chANXA2 were similar to those of the mock group, which indicates that the antibody against ANXA2 could not inhibit ALV-A infection/replication in DF1 cells. These data clearly demonstrate that blocking chANXA2 with a specific antibody can effectively and specially inhibit the infection/replication of ALV-J.

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