Repositioning basic residues in the M domain of the rous sarcoma virus Gagprotein

The first 86 residues of the Rous sarcoma virus (RSV) Gag protein form a me mbrane-binding (M) domain that directs Gag to the plasma membrane during bu dding. Unlike other retroviral Gag proteins, RSV Gag is not myristylated; h owever, the RSV M domain does contain 11 basic residues that could potentia lly interact with acidic phospholipids in the plasma membrane. To investiga te this possibility, we analyzed mutants in which basic residues in the M d omain were replaced with asparagines or glutamines. The data show that neut ralizing as few as two basic residues in the M domain blocked particle rele ase and prevented Gag from localizing to the plasma membrane. Though not as severe, single neutralizations also diminished budding and, when expressed in the context of proviral clones, reduced the ability of RSV to spread in cell cultures. To further explore the role of basic residues in particle p roduction, we added lysines to new positions in the M domain. Using this ap proach, we found that the budding efficiency of RSV Gag can be improved by adding pairs of lysines and that the basic residues in the M domain can be repositioned without affecting particle release. These data provide the fir st gain-of-function evidence for the importance of basic residues in a retr oviral M domain and support a model in which RSV Gag binds to the plasma me mbrane via electrostatic interactions.