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-proteincoupled receptor isolated from osteosarcoma cells. The ability to generate unique envelopes capable of using tissue-or diseasespecific receptors marks an advance in the development of efficient gene-therapy vectors.library screening ͉ retroviral entry ͉ viral envelope ͉ viral receptor

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(11)(12)(13)] viral receptors. With these evolutionary observations, it is of great interest as to whether a given retrovirus must remain restricted to use receptors within the broad class of the original receptor or can be altered to use receptors beyond these protein family boundaries.A variety of approaches have been developed for altering the receptor usage of viral delivery systems. Receptor specificity and binding of the viral Envelope (Env) is mapped to the N-terminal half of the surface (SU) protein through the close interaction of two highly variable regions, VR1 (VRA) and VR2 (VRB) (14-18). Exchange of a defined VR1 segment between the FeLV A and C subgroups altered the viral host range (19). In our studies, codons for 10/11 amino acids within this VR1 FeLV Env receptor-binding region were randomized within a FeLV A/C Env chimera backbone (20). Viral particles bearing this library of Ͼ1 million viral VR1 Env variants are selected for productive infection (20)(21)(22). This library displays the targeting peptide within the context of the authentic Env backbone, eliminating the problem that preidentified phage display peptides inserted into alternative contexts do not maintain their original properties (23). Alternative approaches have involved the addition of targeting domains (24, 25), peptides (23, 26), or single-chain antibodies (27) within the framework of viral Env or capsids (28-30) or through heterologous intermediates (31). Successful retargeting for Sindbis Env pseudotyped onto lentiviral particles has been achieved with the insertion of the Protein A ZZ domain and sandwiching targeting antibodies to the modified particles (32) or through coexpression of Env proteins and surfacebinding molecules (33).Screening the FeLV random Env library on feline AH927 cells identified the A5 Env isolate, which also efficiently infected human 293T embryonic kidney cells (21). Superinfection interference studies indicated that the receptor for A5 was outside the receptor family used by FeLV A, B, and C and MuLV 4070A viruses (21). The A5 Env therefore provided an excellent system to examine the range of proteins capable of serving as receptors for retroviral Env libraries. In this study, the receptor for the A5 Env isolate was identified as the SLC35F2 protein, a transporter protein of unknown function, through screening a feline AH927 cDNA library. ResultsDirected evolution allows for the selection of proteins with new properties. Using this approach, randomization of the FeLV Env receptor-binding domain, followed by selection for productive infection, identified Env isolates with receptor usage outside the interference groups of the known FeLV A, B, and C viruses (21,22). This result implies that the selection of a receptor protein can occur through screening a single round of viral infection. To definitively prove that alternative viral receptors can be selected, the receptor protein for the A5 Env isolate was identified through cDNA screening. The authors declare no conflict of interest.Abbreviations: Env,...

Libraries of feline leukemia virus subgroup A (FeLV-A)-derived envelope (Env) proteins with random peptides incorporated into the cell-targeting region were screened for productive gene delivery to the PC-3 human prostate cell line. In order to increase the efficiency of recovering and testing functional clones, the screen was performed in the presence of a replication-competent 4070A Env-expressing virus under conditions of viral interference. The Env proteins resulting from this library screen were able to mediate gene delivery to 4070A-infected human PC-3, DU145 prostate and TE671 rhabdomyosarcoma cells in the presence, but not absence, of 4070A helper virus. FeLV-A, FeLV-B and Moloney murine leukemia virus (Mo-MuLV) Env proteins were unable to substitute for 4070A Env. Flow cytometry and Western blot analyses indicated increased cell-surface expression and virion incorporation of library-derived Env proteins in the presence of 4070A Env. Interference assays on cells infected with both 4070A and FeLV-B are consistent with the combination of library-derived and 4070A Env proteins utilizing the Pit1 receptor.

BackgroundOsteosarcomas are the most common primary bone malignancies found in children and adolescents. An optimized system was developed for efficient retroviral gene delivery into solid 143B osteosarcoma tumors in mice using a retargeted Env. In these studies, the viral Env CP was isolated from an in vitro screen of a library of feline leukemia virus Env randomized in the receptor-binding domain and maintained high titer on human 143B osteosarcoma cell line.FindingsThe vector developed to express the random Env libraries encoded the drug selectable marker neo. To adapt this for studies in live animals, the murine based vector was modified to express the luciferase gene. The bicistronic vector developed expressed both the CP Env and luciferase in the presence of either the MPMV CTE or a WPRE element. Virus bearing the CP FeLV Env variant maintained high titers after concentration allowing for direct visualization of delivery of the luciferase gene in subcutaneous 143B osteosarcoma tumors.ConclusionThis system serves as a proof-of-concept for the use of novel FeLV Env pseudotyped MLV particles for in vivo gene delivery. Gene delivery and expression of lucerifase from viral particles bearing the CP Env was readily detected in live mice after a single round of intratumor injection.