Selection of feline leukemia virus envelope proteins from a library by functional association with a murine leukemia virus envelope

Abstract

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.

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

“…The introduction of plasmid CeB (46) expressing the gag-pol gene into human TE671 cells producing TECeB [F2 isolate; (47)] and TELCeB6 cells expressing the ⌿ ϩ lacZ gene plus CeB (46) were previously described. The maintenance of the human DU145, human A498, human TE671, TECeB, TELCeB6 cells, human 293T, 293TCeB, and feline AH927 were as previously described (21,22).…”

Library selection and directed evolution approaches to engineering targeted viral vectors

“…Large pools of viral envelope mutants can be generated using a number of approaches, including randomly shuffling gene templates from different parent viruses , inserting random peptide sequences at a specific location Roth, 2002, 2003;Bupp et al, 2005Bupp et al, , 2006, or inserting a defined peptide into random locations Schaffer, 2006a, 2006b). The resulting envelope mutant genetic library is inserted into recombinant viral genomes, and each envelope protein mutant is packaged to generate virus particles encompassing the genome that encodes it.…”

“…Some of the earliest efforts to apply library selection approaches to target retroviral vectors involved libraries where short, random peptide sequences were genetically substituted into the receptor-determining region (RDR) of feline leukemia virus subgroup A (FeLV-A) envelope proteins followed by selection on target cells (Table I) Roth, 2002, 2003;Bupp et al, 2005Bupp et al, , 2006. In contrast to MLV envelope proteins that require changes in both variable regions of their receptor-binding domain (VRA and VRB), modification of only the VRA domains of FeLV envelope proteins allows modulation of viral tropism without severe structural perturbation (Bupp and Roth, 2002).…”

“…Furthermore, a library, whose individual viral particle variants contained/displayed both the same random peptide library as well as amphotropic murine leukemia 4070A virus envelope proteins on their surfaces, yielded an interesting result after selection on 4070A-infected PC-3 human prostate cells. The envelope protein variants that emerged from this selection apparently cooperated with 4070A envelope proteins to infect PC-3 cells, such that preinfection of these cells with wild-type 4070A retrovirus prior to addition of the novel viruses competed away most but not all of the latter's infectivity (Bupp et al, 2006). Both rational ligand insertion and random peptide display require knowledge of insertion sites that do not compromise envelope function and are exposed on the protein surface for receptor binding Roth, 2002, 2003;Bupp et al, 2005Bupp et al, , 2006.…”

“…The envelope protein variants that emerged from this selection apparently cooperated with 4070A envelope proteins to infect PC-3 cells, such that preinfection of these cells with wild-type 4070A retrovirus prior to addition of the novel viruses competed away most but not all of the latter's infectivity (Bupp et al, 2006). Both rational ligand insertion and random peptide display require knowledge of insertion sites that do not compromise envelope function and are exposed on the protein surface for receptor binding Roth, 2002, 2003;Bupp et al, 2005Bupp et al, , 2006. However, if information on such permissive insertion sites is not available, it can be also newly obtained by transposon-based scanning of envelope proteins (Rothenberg et al, 2001;Yu and Schaffer, 2006b).…”