We have previously reported a set of Moloney murine leukemia virus derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insertion of the Pit-2 binding domain (BD) at the N terminus of the ecotropic retroviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2059-2064, 1996). The resulting chimeric envelopes share two BDs: an additional N-terminal BD (Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 BD). By inserting a variety of different amino acid spacers between the two binding domains, we showed that retroviruses can potentially use the targeted cell surface receptor Pit-2, the ecotropic retroviral receptor mCAT-1, or both receptors cooperatively for entry into target cell (S. Valsesia-Wittmann et al., EMBO J 6:1214-1223, 1997). An extreme example of receptor cooperativity was encountered when envelopes with specific proline-rich interdomain spacers (PRO spacers) were tested: both receptors had to be coexpressed at the surface of the targeted cells to cooperatively allow infection. Here, we characterized the role of PRO spacer in the cooperation of receptors. We have shown that the particular organization of the PRO spacera -turn polyprolinewas responsible for the cooperative effect. In the native configuration of the viruses, the structure masked the regions located downstream of the PRO spacer, thus the mCAT-1 BD. After interaction with the targeted Pit-2 receptor, the BD of the backbone envelope became accessible, and we demonstrated that interaction between the mCAT-1 BD and the mCAT-1 receptor is absolutely necessary. This interaction leads to natural fusion triggering and entry of viruses into targeted cells.