Identification of a cellular receptor for subgroup E avian leukosis virus

Abstract

Genetic studies in chickens and receptor interference experiments have indicated that avian leukosis virus (ALV)-E may utilize a cellular receptor related to the receptor for ALV-B and ALV-D. Recently, we cloned CAR1, a tumor necrosis factor receptor (TNFR)-related protein, that serves as a cellular receptor for ALV-B and ALV-D. To determine whether the cellular receptor for ALV-E is a CAR1-like protein, a cDNA library was made from turkey embryo fibroblasts (TEFs), which are susceptible to ALV-E infection, but not to infection by ALV-B and ALV-D. The cDNA library was screened with a radioactively labeled CAR1 cDNA probe, and clones that hybridized with the probe were isolated. A 2.3-kb cDNA clone was identified that conferred susceptibility to ALV-E infection, but not to ALV-B infection, when expressed in transfected human 293 cells. The functional cDNA clone is predicted to encode a 368 amino acid protein with significant amino acid similarity to CAR1. Like CAR1, the TEF protein is predicted to have two extracellular TNFR-like cysteine-rich domains and a putative death domain similar to those of TNFR I and Fas. Flow cytometric analysis and immunoprecipitation experiments demonstrated specific binding between the TEF CAR1-related protein and an immunoadhesin composed of the surface (SU) envelope protein of subgroup E (RAV-0) virus fused to the constant region of a rabbit immunoglobulin. These two activities of the TEF CAR1-related protein, specific binding to ALV-E SU and permitting entry only of ALV-E, have unambiguously identified this protein as a cellular receptor specific for subgroup E ALV.

Figure 1

The pBKTEF24 plasmid specifically confers susceptibility to ALV-E infection when transfected in human 293 cells. The 293 cells were transiently transfected with plasmid pBKTEF24, plasmid pBK7.6–2 encoding CAR1 (5), or no DNA (mock). The transfected cells were challenged with either a subgroup E virus, RCASBP(E)/AP (13) (A), or a subgroup B virus, RCASBP(B)/AP (B). Three days after viral challenge, cells were stained to identify those that expressed the virally encoded heat-stable AP, and the stained foci were counted. The data shown represent the average number of foci obtained in three independent experiments.

Figure 2

The pBKTEF24 clone encodes a type I transmembrane protein. The complete DNA sequence of the pBKTEF24 cDNA clone is shown with the predicted amino acid sequence of the protein product. The putative transmembrane domain is underlined and the three potential N-linked glycosylation sites (N-X-S/T) are boxed. The predicted leader peptidase cleavage site (15) is marked with an arrow.

Figure 3

The TEF protein is highly related to CAR1. The amino acid sequence of the TEF protein (TEF24) is aligned with that of CAR1. Identical amino acids are marked with a dash in the CAR1 sequence, and gaps in either sequence are indicated by spaces. The CRDs, which are discussed in the text, are marked with hatched bars. The putative death domains of both proteins are marked by a shaded bar.

Figure 4

The TEF protein bound specifically to a subgroup E SU-immunoadhesin. Protein A-Sepharose beads bound to either SUB or SUE immunoadhesin were incubated with protein lysates from 293 cells transfected with either plasmid pBKTEF24 or plasmid pBK7.6–2 expressing CAR1 or with no DNA (mock). The immunoprecipitated protein was subjected to SDS/polyacrylamide gel electrophoresis and immunoblotted with the IQS579 CAR1 peptide antibody.

Figure 5

The TEF protein is expressed on the cell surface and binds specifically to an ALV-E SU immunoadhesin. Human 293 cells transfected with plasmid pBKTEF24 or with no DNA (Mock) were bound to either SUB-rIgG (A) or SUE-rIgG (B), and flow cytometric analysis was performed as described previously (10). The pBKTEF24-expressing cells were specifically bound by SUE-rIgG but not by SUB-rIgG.