Identification of an Extracellular Domain within the Human PiT2 Receptor That Is Required for Amphotropic Murine Leukemia Virus Binding

PiT1 and PiT2 are type III sodium-dependent phosphate transporters that also function as receptors for the mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and amphotropic murine leukemia virus (A-MuLV), respectively (10, 18, 19, 34, 36). While these receptors have similar cellular functions and structures, they do not overlap in their virus receptor functions; this has been attributed to critical amino acid differences between PiT1 and PiT2. Early structural predictions for the arrangement of the PiT receptors in the plasma membrane were based on Kyte-Doolittle hydropathy analyses (8). Both proteins were initially predicted to be nearly identical in structure, each comprising 10 transmembrane (TM) domains. Additionally, the observed absence of a signal peptide for both proteins was used to assign cytoplasmic locations for the N and C termini; both were initially predicted to contain five extracellular domains (ECDs) and four intracellular domains, with all potential N-linked glycosylation sites being positioned within intracellular domains (8). In order to understand how differences in amino acid composition between PiT1 and PiT2 affect receptor function, researchers have used chimeric PiT1-PiT2 proteins to map regions that are critical for GALV (2, 5, 9, 21, 22, 27, 32) and A-MuLV (12, 13, 14, 21, 28, 30) entry. Previous studies based on Kyte-Doolittle hydropathy models of PiT1 and PiT2 have demonstrated that replacement of the second ECD (ECD2) of PiT1 with the corresponding region of PiT2 results in a chimeric protein which functions as an A-MuLV receptor (12). This result was supported by studies by Lundorf et al. that showed that substitution of PiT2 residues from ECD2 and flanking regions for the corresponding residues of Pho-4, a sodium-dependent phosphate transporter from the filamentous fungus Neurospora crassa, confers A-MuLV receptor function to Pho-4 (13). It has recently been experimentally determined that both the N and C termini of PiT2 are extracellular, thus reorienting the former first, second, and third ECDs to the cytosol and the former first and second intracellular domains to extracellular positions (26). The reorientation of the N-terminal third of the PiT2 protein was further validated by experiments demonstrating that PiT2 is a glycoprotein carrying an N-linked oligosaccharide in the more recently predicted ECD1 (26). It should be noted that chimeric PiT1-PiT2 receptor studies implicating the former ECD2 as critical to A-MuLV infectivity utilized the earlier Kyte-Doolittle-based PiT receptor topology. The current topological model positions the region formerly designated ECD2 in the cytosol. Interestingly, the region currently designated ECD1 was still present in these chimeras, although it was unclear at the time that this domain was extracellular. Thus, previous results may have been misinterpreted to implicate the former ECD2 as being important for PiT2-mediated A-MuLV infection when, in fact, the receptor function was being mediated by what is now called ECD1. For A-MuLV receptor function studies, CHOK1 cells have been the preferred cell line, based on the observation that CHOK1 cells are resistant to infection by both GALV and A-MuLV. It was suggested that the reason these cells are refractory to A-MuLV infection is because they express nonfunctional receptors or receptors masked by a tunicamycin-sensitive inhibitor (3, 16, 17, 35). However, it was shown more recently that simply overexpressing the endogenous PiT2 receptor in CHOK1 cells (PiT2CHO) resulted in susceptibility to both GALV and A-MuLV, while expression of the endogenous PiT1 receptor (PiT1CHO) resulted in susceptibility to GALV (30). These findings suggest that the block to A-MuLV infection of CHOK1 cells is not due to the absence of functional receptors, and therefore, CHOK1 cells may not be the best system for investigating A-MuLV receptor function. As stated above, studies attempting to identify regions of PiT2 that are important for A-MuLV entry used virus infectivity assays to measure receptor function. None of these studies explored the binding capability of A-MuLV to various chimeric receptors, raising the possibility that certain nonfunctional A-MuLV receptors retained the ability to bind A-MuLV while not facilitating entry into target cells. Therefore, while previous studies were useful for mapping receptor regions important for A-MuLV entry, regions of the receptor that are critical for A-MuLV virus binding were not directly assessed. The purpose of this study was to map the region of PiT2 required for A-MuLV binding and/or entry into host cells, using chimeric PiT1-PiT2 receptors based on the experimentally validated topological models of PiT2, proposed by Salaun and coworkers (25, 26), and PiT1, proposed by Farrell et al. (6). Herein we report that the first ECD of PiT2 is critical for A-MuLV binding and entry.