Amphotropic Envelope/Receptor Interactions

Abstract

The amino-terminal domain of murine leukemia virus (MuLV) envelope glycoproteins (SU) is sufficient for binding cell surface receptors and mediating entry into cells. This domain is an anti-parallel ß sandwich with two helical subdomains forming loops adjacent to the ß-sandwich. The loops contains the determinants involved in receptor recognition. A purified 208 aminoacid fragment containing the amphotropic receptor binding domain competes with the binding of amphotropic particles and inhibits the entry of amphotropic retrovirus vectors. Concentrations inhibiting entry appeared much lower than that required to abolish binding. This suggested that only a fraction of the receptors are competent for processing retrovirus entry. Requirement for cell factors or for association with specific cell structure may account for this restriction. Alternatively, the association of several receptor molecules may be required for processing entry. Scatchard analysis performed with ¹²⁵I-labeled AS208 showed curvilinear plots with downward concavity, indicating that receptor cooperativity participates in binding efficiency.

The amphotropic receptor is the Pit-2 molecule encoded by the ram-1 gene. It is a multiple transmembrane protein which functions as a phosphate/Na symporter. Sequence analysis predict 10 transmembrane domains, 5 extracellular loops and intracellular N- and C-terminal extremities. We inserted all amino acid epitope of the VSV G protein at various locations including the extracellular loop 5 and the C-terminal extremity. The tagged receptors were expressed in CHO cells which do not express the amphotropic receptor naturally. Virus particle binding and infection mediated by tagged or wild type receptors were equivalent. Axiti-VSV-G mAbs immunoprecipitated a 70kDa glycosylated receptor molecules in transfected cells. Flowcytometry and immunofluorescence analysis revealed that, in contrast with the predicted topology, the C-terminal epitope is extracellular. In naive cells, the signal was homogeneously spread over the plasma membrane. After one hour incubation with virus particles, the signal condensed as large granulations. This observation was consistent with the hypothesis of a clustering of the amphotropic receptor in response to particle binding. Spatial reorganization of the receptors was observed in response to phosphate. High phosphate concentration induced spreading of the receptor and inhibited virus entry. In contrast, phosphate starvation induced receptor aggregation, induced pictures evoking stress cable formation and membrane ruffling, and increased virus entry. Actin staining confirmed colocalization of cell surface receptors with intracellular actin filaments. Colocalization was still observed after Cytochalasine D treatment, which disrupt actin network. Virus entry was completely abolished in the presence of cytochalasine. These data show that association of the amphotropic receptor with cytoskeleton structures plays a crucial role in virus entry. We are currently investigating the signalisation pathways stimulated by virus particle binding which induce receptor reorganisation and allow for virus entry into cells.