Identification of a retroviral receptor used by an envelope protein derived by peptide library screening

Abstract

This study demonstrates the power of a genetic selection to identify a variant virus that uses a new retroviral receptor protein. We screened a random peptide library within the receptor-binding domain of a feline leukemia virus retroviral Envelope (FeLV Env) protein for productive infection of feline AH927 cells. One variant, A5, obtained with altered tropic properties acquired the ability to use the solute carrier protein family 35 member F2 (SLC35F2) as a receptor. The SLC35F2 protein is a presumed transporter of unknown function predicted to encode 8 to 10 transmembrane-spanning regions and is not homologous to any identified retroviral receptor. Expression of the feline SLC35F2 cDNA in nonpermissive cells renders the cells susceptible to infection by A5 virus, with remarkably high titers in the range of 10(5) infectious units per ml. The human SLC35F2 ORF also functioned as the retroviral receptor, albeit at lower efficiency than the feline homologue. The successful selection of a novel molecule, the SLC35F2 transporter/channel-type protein, as a receptor by the FeLV Env backbone suggests that multipass transmembrane proteins may be particularly suited for use in productive viral entry and fusion. The analysis of retroviral Env libraries randomized in the receptor-binding domain offers a viable means to develop viral vectors targeted to specific cell types in the absence of known targeting ligands.

Fig. 1.

Schematic of cDNA library screen. The individual steps involved in screening the retroviral cDNA expression library, including the A5 viral challenges, are outlined.

Fig. 2.

Comparison of feline and human SLC35F2 homologues. (Upper) Schematic comparison of the feline and human SLC35F2 cDNAs, including the ORF and 3′ untranslated region (3′UTR). (Lower) Protein sequence alignment of the predicted feline and human SLC35F2 proteins. The human protein ID is AAH39195.1 (sequence accession no. BC039195). Amino acid identities are marked by dots. Areas shaded in gray correspond to central transmembrane helix segments predicted by using the MEMSTAT3 program with an N-terminal inside loop (34).

Fig. 3.

PCR analysis of the primary and secondary cDNA isolates. Primers within the feline SLC35F2 ORF (42fs and 42–17ext) were used to detect the feline SLC35F2 cDNA within primary and secondary isolates. The arrow marks the position of the predicted 538-bp product. Control cells (nonpermissive A498) lacking cDNA library and PCR performed in the absence of genomic template DNA are included. M, marker DNA. The predicted molecular weights are indicated at the right.

Fig. 4.

Introduction of feline SLC35F2 cDNA renders cells permissive to A5 infection. Feline SLC35F2 cDNA was introduced into nonpermissive cells by retroviral infection of VSV-G-pseudotyped particles packaging the pMX1/42–17 cDNA. All cells are challenged with A5/lacZ virus. (A) Nonpermissive A498 cells. (B) A498 cells expressing feline SLC35F2 cDNA. (C) DU145 cells. (D) DU145 expressing feline SLC35F2 cDNA. Titers of A5/lacZ virus (lsu per ml of virus) are indicated in each box.

Fig. 5.

Expression of SLC35F2 on the cell surface. A5 virus binding was detected by flow-cytometric analysis using the C11D8 mAb recognizing the FeLV Env backbone (36). Data are representative of six independent experiments. (A) A5 virus binding to human A498 cells. Green, nonpermissive A498 cells; red, A498 cells expressing the pMX1/felineSLC35F2 construct; black, A498 cells expressing the pMX1/human SLC35F2 construct. (B) Binding to permissive feline AH927 cells. Blue, control assay performed in the absence of C11D8 antibody; green, control binding performed in the absence of virus; red, complete binding reaction performed with A5 virus plus C11D8 antibody.