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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) Infection of the chANXA2-transfected cells.Geese embryo fibroblasts (GEF) and 293T cells were transfected with chANXA2, and the transfected cells were then infected with ALV-J at an MOI of 5. (A), The replication of ALV-J in the 293T cells transfected with ANXA2 was measured by real-time PCR; (B), The replication of ALV-J in the GEF cells transfected with ANXA2 was measured IFA after inoculating the homogenate from the transfected GEF cells to the DF1 cells.
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f4: (Qin) Infection of the chANXA2-transfected cells.Geese embryo fibroblasts (GEF) and 293T cells were transfected with chANXA2, and the transfected cells were then infected with ALV-J at an MOI of 5. (A), The replication of ALV-J in the 293T cells transfected with ANXA2 was measured by real-time PCR; (B), The replication of ALV-J in the GEF cells transfected with ANXA2 was measured IFA after inoculating the homogenate from the transfected GEF cells to the DF1 cells.

Mentions: To extend our finding and investigate whether the over-expression of the chANXA2 protein in ALV-J non-permissible cells could induce susceptibility to ALV-J infection, the replication of ALV-J in the 293T cells transfected with chANXA2 was analysed by IFA and real-time PCR. Real-time PCR revealed that the relative expression of the ALV-J envelope gene was increased in the chANXA2-transfected 293T cells at 72 h post-infection (Fig. 4A). However, the 293T cells that were transfected with chANXA2 exhibited no visible specific fluorescence with the JE9 antibody to ALV-J Env. These data indicate that ALV-J could enter the 293T cells that were over-expressing chANXA2, but the viral replication of the ALV-J was restricted in the transfected 293T cells. To further confirm this finding, we also transfected chANXA2 into goose embryo fibroblast (GEF) cells in which the ALV-J could not grow. After 48 h, the transfected GEFs were infected with ALV-J. The GEF cells were maintained in DMEM medium for 2 days. Next, the mixture of GEF cells and supernatants were inoculated into fresh DF1 cells. Interestingly, as shown in Fig. 4B, virus was recovered from the GEF cells transfected with chANXA2, but no virus was obtained from the GEF cells that were transfected with the control plasmid. Together, these data demonstrate that the over-expression of chANXA2 in 293T or GEF cells can support ALV-J entry into these ALV-J non-permissible cells.


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) Infection of the chANXA2-transfected cells.Geese embryo fibroblasts (GEF) and 293T cells were transfected with chANXA2, and the transfected cells were then infected with ALV-J at an MOI of 5. (A), The replication of ALV-J in the 293T cells transfected with ANXA2 was measured by real-time PCR; (B), The replication of ALV-J in the GEF cells transfected with ANXA2 was measured IFA after inoculating the homogenate from the transfected GEF cells to the DF1 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: (Qin) Infection of the chANXA2-transfected cells.Geese embryo fibroblasts (GEF) and 293T cells were transfected with chANXA2, and the transfected cells were then infected with ALV-J at an MOI of 5. (A), The replication of ALV-J in the 293T cells transfected with ANXA2 was measured by real-time PCR; (B), The replication of ALV-J in the GEF cells transfected with ANXA2 was measured IFA after inoculating the homogenate from the transfected GEF cells to the DF1 cells.
Mentions: To extend our finding and investigate whether the over-expression of the chANXA2 protein in ALV-J non-permissible cells could induce susceptibility to ALV-J infection, the replication of ALV-J in the 293T cells transfected with chANXA2 was analysed by IFA and real-time PCR. Real-time PCR revealed that the relative expression of the ALV-J envelope gene was increased in the chANXA2-transfected 293T cells at 72 h post-infection (Fig. 4A). However, the 293T cells that were transfected with chANXA2 exhibited no visible specific fluorescence with the JE9 antibody to ALV-J Env. These data indicate that ALV-J could enter the 293T cells that were over-expressing chANXA2, but the viral replication of the ALV-J was restricted in the transfected 293T cells. To further confirm this finding, we also transfected chANXA2 into goose embryo fibroblast (GEF) cells in which the ALV-J could not grow. After 48 h, the transfected GEFs were infected with ALV-J. The GEF cells were maintained in DMEM medium for 2 days. Next, the mixture of GEF cells and supernatants were inoculated into fresh DF1 cells. Interestingly, as shown in Fig. 4B, virus was recovered from the GEF cells transfected with chANXA2, but no virus was obtained from the GEF cells that were transfected with the control plasmid. Together, these data demonstrate that the over-expression of chANXA2 in 293T or GEF cells can support ALV-J entry into these ALV-J non-permissible cells.

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