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. 2000 Apr;74(8):3572-78.
doi: 10.1128/jvi.74.8.3572-3578.2000.

Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry

H B Adkins  1 , J Brojatsch, J A Young
Affiliations

Identification and characterization of a shared TNFR-related receptor for subgroup B, D, and E avian leukosis viruses reveal cysteine residues required specifically for subgroup E viral entry

H B Adkins et al. J Virol. 2000 Apr.

Abstract

Genetic and receptor interference data have indicated the presence of one or more cellular receptors for subgroup B, D, and E avian leukosis viruses (ALV) encoded by the s1 allele of the chicken tvb locus. Despite the prediction that these viruses use the same receptor, they exhibit a nonreciprocal receptor interference pattern: ALV-B and ALV-D can interfere with infection by all three viral subgroups, but ALV-E only interferes with infection by subgroup E viruses. We identified a tvb(s1) cDNA clone which encodes a tumor necrosis factor receptor-related receptor for ALV-B, -D, and -E. The nonreciprocal receptor interference pattern was reconstituted in transfected human 293 cells by coexpressing the cloned receptor with the envelope (Env) proteins of either ALV-B or ALV-E. This pattern of interference was also observed when soluble ALV surface (SU)-immunoglobulin fusion proteins were bound to this cellular receptor before viral challenge. These data demonstrate that viral Env-receptor interactions can account for the nonreciprocal interference between ALV subgroups B, D, and E. Furthermore, they indicate that a single chicken gene located at tvb(s1) encodes receptors for these three viral subgroups. The TVB(S1) protein differs exclusively at residue 62 from the published subgroup B- and D-specific receptor, encoded by the s3 allele of tvb. Residue 62 is a cysteine in TVB(S1) but is a serine in TVB(S3), giving TVB(S1) an even number of cysteines in the extracellular domain. We present evidence for a disulfide bond requirement in TVB(S1) for ALV-E infection but not for ALV-B infection. Thus, ALV-B and ALV-E interact in fundamentally different ways with this shared receptor, a finding that may account for the observed biological differences between these two ALV subgroups.

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Figures

FIG. 1
FIG. 1
The pBK3-1 clone encodes a receptor for ALV-B and ALV-E. (A) Human 293 cells were transfected with plasmid pBK3-1 (293:pBK3-1) or no DNA (293) and were then challenged with subgroup B-specific (RCASH-B) or subgroup E-specific (RCASH-E) ALV vectors encoding hygromycin B phosphotransferase. The resultant hygromycin B-resistant colonies were counted, and the data shown represent the average numbers obtained in three independent experiments. (B) Human 293 cells transfected with plasmid pBK3-1 or with no DNA were lysed in NP-40 lysis buffer and subjected to immunoprecipitation with subgroup B-specific (SUB-rIgG) or subgroup E-specific (SUE-rIgG) SU-rabbit Ig fusion proteins. The immunoprecipitated proteins were then subjected to immunoblotting using an antiserum specific for TVB (1) and visualized by enhanced chemiluminescence. (C) The death domain of TVBS1 is not required for cell surface expression or binding to ALV-B or ALV-E SU. Transfected 293 cells expressing TVBS1(ΔDD), a truncated receptor lacking the cytoplasmic death domain, were incubated with SUB-rIgG or SUE-rIgG and with a fluorescein isothiocyanate-conjugated antibody specific for rabbit Igs. The cells were then analyzed by flow cytometry.
FIG. 2
FIG. 2
Reconstitution of nonreciprocal interference between ALV-B and ALV-E with the cloned TVBS1 protein. (A) Four independent lines of human 293 cells stably expressing TVBS1(ΔDD) and EnvE (EnvE-S1 cells) were challenged with 10 μl (approximately 103 infectious units) of MLV-lacZ (EnvB) or with 100 μl (approximately 104 infectious units) of MLV-lacZ (EnvE). The numbers of infected β-galactosidase-positive cells obtained in a representative experiment were determined, and these numbers were corrected so that they represent those that would be obtained per milliliter of each original virus stock. (B) Human 293 cells transiently expressing the full-length TVBS1 protein were incubated with extracellular supernatants containing SUB-rIgG or SUE-rIgG or with a control supernatant lacking any SU-rIgG protein (no block) for 1 h at 37°C prior to the addition of either RCASH-B or RCASH-E viruses. After approximately 2 weeks of selection in medium containing hygromycin B, the drug-resistant colonies were counted. The data shown represent the average number of colonies obtained in three independent experiments.
FIG. 3
FIG. 3
Mutational analysis of the first four cysteine residues of TVBS1. (A) TVBS3 and TVBS1 differ by a serine-to-cysteine substitution at residue 62 (shaded). The regions of TVBS1, TVBS3, and TVBT that encompass all extracellular cysteine residues (amino acids 45 to 144) are shown aligned, and the predicted intrachain disulfide bonds in TVBS1 are indicated. (B) All of the mutant receptors bearing cysteine mutations were expressed at the cell surface and bind to SUB-rIgG. Human 293 cells transfected with no DNA or with plasmids encoding wild-type TVBS1(ΔDD) (WT) or with mutant forms of this receptor were incubated with extracellular supernatant containing SUB-rIgG and with a fluorescein isothiocyanate-conjugated antibody specific for rabbit Igs. The cells were then analyzed by flow cytometry; the results shown were obtained with nonreceptor-expressing cells (open histograms) and receptor-expressing cells (shaded histograms).
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