Identification of the Segments of the Mouse Transferrin Receptor 1 Required for Mouse Mammary Tumor Virus Infection

Abstract

Most enveloped viruses enter cells through binding of virion surface envelope proteins to receptors found on the plasma membrane of the cell. The beta retrovirus mouse mammary tumor virus (MMTV) uses transferrin receptor 1 (TfR1) to enter cells in a pHdependent mechanism, probably co-trafficking with TfR1 to an acidic compartment where virus entry occurs. We have shown here that, although mouse and rat TfR1 function as entry receptors, cat, dog, hamster, or human TfR1s do not support MMTV infection. We also demonstrated that MMTV entry is independent of transferrin, iron, and the TfR1 cofactor hereditary hematochromatosis HFE protein. Using chimeric mouse/human hybrid TfR1 constructs, we determined the site of interaction with MMTV and found that it maps to two segments physically disparate from the TfR and HFE binding sites. Thus, MMTV has apparently evolved to enter cells independently of the iron status of the host.Much is known about the role of transferrin receptor 1 (TfR1) 2 and its accessory proteins in the uptake of iron into cells. Iron-bound transferrin (Tf) first binds to TfR1 at neutral pH at the cell surface and traffics with the receptor to an acidic compartment where iron is released and transported to the cytoplasm via divalent metal transport protein 1 (reviewed in Ref. 1). The receptor then recycles back to the cell surface, where iron-free Tf is released and a new molecule of iron-bound Tf binds. At least one additional cell surface protein has been implicated in the process of iron uptake, the ␤2-globulin-associated, major histocompatibility locus-encoded hereditary hematochromatosis HFE protein, which competes with Tf for binding to TfR1 and is thought to limit iron uptake by cells in which it is co-expressed with the receptor (1). Indeed, mice with targeted deletion of the ␤2-globulin gene develop an iron overload disorder similar to that seen in HFE patients (2). HFE protein is found in a number of cell types, including epithelia, fibroblasts, and monocytes/macrophages (3, 4), and transcripts encoding the gene are present in many tissues, including those infected by MMTV in vivo such as lymphoid and mammary gland (GNF Symatlas (symatlas. gnf.org/SymAtlas/) (5) and Unigene (www.ncbi.nlm.nih.gov/UniGene)).The crystal structure of the human TfR1 ectodomain has been solved, both as a homodimer (6) and in complex with the HFE protein (7). The protein is defined by three domains, a protease-like domain (residues 122-188 and 384 -606) with similarity to carboxyl-and amino-peptidases but no apparent enzymatic activity, an apical domain (residues 189 -383), and a helical domain (residues 607-760) required for subunit dimerization. The solution of the structure in combination with mutagenesis studies (8 -11) and cryoelectron microscopy (12) have defined overlapping and competing binding sites for Tf and HFE located in the helical region of the protein. Sequence comparison of this region from different species shows strong conservation of an RGD-like sequence that is critical for Tf and HFE binding (...

New World Clade B Arenaviruses Can Use Transferrin Receptor 1 (TfR1)-Dependent and -Independent Entry Pathways, and Glycoproteins from Human Pathogenic Strains Are Associated with the Use of TfR1

“…It has previously been reported that mouse mammary tumor virus (MMTV) uses TfR1 for entry and, in an opposite pattern from what we observe for the arenaviruses, is able to use the murine but not the human version of this molecule (34). Through the use of human/murine chimeras, the residues responsible for this species difference in MMTV utilization have been mapped to two regions that are spatially close on the surface of TfR1, at the outer edge of the TfR1 homodimer, in a region that is distinct from the binding sites for either of its natural ligands, transferrin or the hereditary hematochromatosis protein, HFE (34). Since it has also been demonstrated that soluble transferrin does not compete with MACV GP vectors for entry into human cells (25), we asked if the clade B GPs could be binding to a region of hTFR1 similar to the one MMTV uses on mTfR1.…”

“…We analyzed the ability of the vectors to enter CHO-K1 cells transfected with a panel of reciprocal chimeras between hTfR1 and mTfR1 (Fig. 7A), with chimeras 3 to 5 and 16 containing substitutions in hTfR1 of the murine sequences necessary to confer full MMTV entry (34). As controls, we used VSV-G and TCRV GP vectors, neither of which is affected by the expression of either hTfR1 or mTfR1 in CHO-K1 cells (Fig.…”

Infectious Entry Pathway Mediated by the Human Endogenous Retrovirus K Envelope Protein

“…Beta-type ENVs are known to have restricted species tropism based on receptor usage; however, the range of cell types tested has been limited to those in which specific retroviruses can replicate (19,(22)(23)(24)(25)(26). Since the VSV core can replicate in all eukaryotic cells in culture, VSV-HERVK provides a unique reagent to interrogate more broadly the ability of HERV-K ENV to mediate entry into those cells.…”

“…MMTV uses the transferrin receptor of certain species of mice and rats (19,(22)(23)(24). JSRV uses Hyal2 from a variety of mammals, but not rodents (25), whereas ENTV uses only Hyal2 of ovine cells (25,26).…”

Receptor determinants of zoonotic transmission of New World hemorrhagic fever arenaviruses

“…2A) for their ability to bind MACV GP1⌬-Fc and support MACV, JUNV, and GTOV GP-mediated entry. Chimeras 1-6 have been previously described (42). All receptor chimeras were expressed on the cell surface to similar levels as determined by flow cytometry (Fig.…”