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

Abstract

(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,...

Pseudotyped Lentiviral Vectors: One Vector, Many Guises

“…Through this approach, FeLV mutants with altered tropism were identified. [82][83][84] A more directed approach for modifying the tropism of envelopes takes advantage of specific ligand-receptor reactions. Such a ligand-based approach was demonstrated by incorporating a protein called heregulin in Mo-MLV virus envelope glycoprotein.…”

Role of Cysteines in Stabilizing the Randomized Receptor Binding Domains within Feline Leukemia Virus Envelope Proteins

“…Our studies have shown that viral gene delivery can be altered using alternative host proteins as target receptors by randomizing the receptor binding domains within the gammaretroviral Env (18)(19)(20)(21)(22)(23)25). Previous techniques produced a limited number of high-affinity Env isolates in each round of screening (18)(19)(20)(21)(22).…”

“…For two of these isolates, A5 and constrained peptide (CP), the corresponding receptors have been identified (22,23). The receptor for the A5 isolate is the SLC35F2 protein, a multitransmembrane spanning protein.…”

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

“…Through randomizing a limited primary sequence within this VRA region, a library of Env proteins have been generated and screened for productive infection into target cells. Through this process, it has been established that retroviral entry can be targeted to novel host-cell receptors (7,8,10). To understand the scope of the potential receptor usage and the correlation with the evolution of known endogenous and exogenous retroviruses, a new FeLV library was generated randomizing 11 amino acids within the VRA region.…”