Single-round selection yields a unique retroviral envelope utilizing GPR172A as its host receptor

Abstract

The recognition by a viral envelope of its cognate host-cell receptor is the initial critical step in defining the viral host-range and tissue specificity. This study combines a single-round of selection of a random envelope library with a parallel cDNA screen for receptor function to identify a distinct retroviral envelope/receptor pair. The 11-aa targeting domain of the modified feline leukemia virus envelope consists of a constrained peptide. Critical to the binding of the constrained peptide envelope to its cellular receptor are a pair of internal cysteines and an essential Trp required for maintenance of titers >10(5) lacZ staining units per milliliter. The receptor used for viral entry is the human GPR172A protein, a G-protein-coupled receptor isolated from osteosarcoma cells. The ability to generate unique envelopes capable of using tissue- or disease-specific receptors marks an advance in the development of efficient gene-therapy vectors.

Fig. 1.

Schematic of the Env library vector and CP-targeting peptide. (A) Schematic of the randomized Env-IRES-puro (RIP) library vector. The SV40 promoter and the neomycin selection marker from pRVL (7) were replaced by an expression cassette in which an internal ribosomal entry site (IRES) drives the expression of puro. Position of the LTRs and viral genes are marked; Ψ, packaging signal. (B) Sequence of the Env isolated from Caki-1. The 11-aa encoding the CP new binding insert are numbered in bold and flanked by the conserved regions of the FeLV envelope. The putative cystine bonds are represented and alternative configurations of bonds between cysteines are possible. The cysteine loop in the parental FeLV Env consists of the outer residues.

Fig. 2.

Titer of the CP Env mutants. The assay is quantifying the transfer of the lacZ marker via wild-type CP or mutant CP Envs. The figure represents the average of the 3 values, with error bars representing 1 SD. Mutations are numbered according to their position in the randomized region of the wild-type CP insert.

Fig. 3.

Binding studies of virus bearing mutant CP on human 143B osteosarcoma cells. Virus-binding to 143B cells was detected by flow-cytometric analysis using the C11D8 mAb recognizing the FeLV Env backbone and goat anti-mouse IgG conjugated with FITC. For each panel, the black line represents the control assay performed in the absence of virus and the red line represents the binding of the wild-type CP virus to the 143B cells. The green line represents the virus bearing the mutated CP Env as indicated on each panel.

Fig. 4.

Schematic diagram of the GPR172A mRNA from 143B cells. The GPR172A gene is encoded on chromosome 8 at position q24.3 (top line). Shown is the 5′ terminus of the gene including the 5′ UTR, with the Met start encoded at position 731 within the gene. Multiple mRNA start sites and splice junctions have been reported within the 5′ UTR for the GPR172A mRNA and are individually listed. Key positions with respect to the transcription start site in the chromosome, as well as the sequence of the 5′ terminus of the mRNAs, are indicated. The 2 cDNAs isolated from 143B cells are shown in comparison with the reported GPR172A mRNAs.

Fig. 5.

GPR172A functions as CP Env viral receptor. (A) Introduction of GPR172A cDNA renders cells permissive to CP infection. Human GPR172A cDNA was introduced into the nonpermissive AH927 cells and UMR-106 cells by retroviral infection (see Materials and Methods). Cells were challenged with virus bearing the CP Env and packaging lacZ. Titers of CP/lacZ virus (LSU/ml) are indicated: nonpermissive cells (light gray); GPR172A expressed in nonpermissive cells (Gray); control 143B permissive cells (black). (B) Introduction of shRNA against GPR172A in permissive 143B cells. VSV-G pseudotyped lentiviral vectors packaging shRNA were used to infected 143B cells, selected for puro^r and challenged with virus bearing the CP Env and packaging lacZ. Viral titer on 143B was 3.0 × 10⁵ LSU/ml. Relative rates of infectivity are shown for shRNAs 11421 and 8272, as well as for the empty vector control (pLKO.1) compared to that of wild-type 143B cells. The figures represent the average of the 3 values with error bars representing 1 SD.