Role of chimeric murine leukemia virus env beta-turn polyproline spacers in receptor cooperation

We have previously reported a set of Moloney murine leukemia virus derived envelopes retargeted to the Pit-2 phosphate transporter molecule, by insert ion of the Pit-2 binding domain (BD) at the N terminus of the ecotropic ret roviral envelope glycoproteins (S. Valsesia-Wittmann et al., J. Virol. 70:2 059-2064, 1996). The resulting chimeric envelopes share two BDs: an additio nal N-terminal BD (Pit-2 BD) and the BD of the ecotropic envelope (mCAT-1 B D). By inserting a variety of different amino acid spacers between the two binding domains, we showed that retroviruses can potentially use the target ed cell surface receptor Pit-2, the ecotropic retroviral receptor mCAT-1, o r both receptors cooperatively for entry into target cell (S. Valsesia-Witt mann et al., EMBO J 6:1214-1223, 1997). An extreme example of receptor coop erativity was encountered when envelopes with specific proline-rich interdo main spacers (PRO spacers) were tested: both receptors had to be coexpresse d at the surface of the targeted cells to cooperatively allow infection. He re, we characterized the role of PRO spacer in the cooperation of receptors . We have shown that the particular organization of the PRO spacer-a P-turn polyproline-was responsible for the cooperative effect. In the native conf iguration of the viruses, the structure masked the regions located downstre am of the PRO spacer, thus the mCAT-1 BD. After interaction with the target ed Pit-2 receptor, the BD of the backbone envelope became accessible, and w e demonstrated that interaction between the mCAT-1 BD and the mCAT-1 recept or is absolutely necessary. This interaction leads to natural fusion trigge ring and entry of viruses into targeted cells.