Bucy, J. located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of RAD50 the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of 10058-F4 the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the 10058-F4 HR-B region contribute jointly to controlling F conformational stability. Enveloped viruses, such as retroviruses, paramyxoviruses, orthomyxoviruses, and filoviruses, infect cells through fusion of their lipid envelope with the plasma membrane or intracellular membranes of the target cell (17, 30). For members of these viral families, membrane merger is mediated by homotrimeric type I fusogenic membrane glycoproteins (FMGs), integral membrane proteins displayed on the surfaces of the viral particles (17, 54). All type I FMGs contain an internal hydrophobic domain of approximately 25 amino acids, generally termed the fusion peptide. Proteolytic cleavage at a specific site yields a metastable native FMG that consists of a transmembrane and a membrane-distal subunit. Subsequent activation of the FMG results in insertion of the fusion peptide, which is located in the transmembrane subunit, into the target membrane (21). Depending on the origin of the FMG, activation can be realized at neutral pH, as postulated, for example, for lentiviruses (4, 27) and most paramyxoviruses, including measles virus (MV) (19), or at low-pH conditions in an endosomal compartment of the target cell, as exemplified by influenza virus (54). Insertion of the fusion peptide into the target membrane is then followed by conformational rearrangements of the FMG trimer that bring the fusion peptide and the transmembrane domain, and hence the target and donor membranes, into close proximity (1, 2, 37, 40, 50, 61), ultimately resulting in the formation of a fusion pore. Instrumental in this process are two highly conserved 4-3 heptad repeat (HR) sequences, one of which is located adjacent to the fusion peptide and near the N terminus of the protein (hence termed the HR-N or HR-A domain), while the other is adjacent to the transmembrane domain and near the C terminus (HR-C or HR-B) (17, 29). Activation of the native FMG and insertion of the fusion peptide into the target membrane are thought to be followed by refolding into a transient hairpin intermediate and the formation of a stable six-helix bundle (6-HB) fusion core structure (17, 54). Analysis of this core structure of lentivirus (7) and paramyxovirus (1, 61) FMGs has revealed a central homotrimeric coiled coil formed by HR-A domains that is surrounded by three HR-B helices in an antiparallel fashion (17, 54). In this model, the process of protein refolding and 6-HB formation is thus coupled to membrane fusion (15, 37, 50). The conformational changes may in fact liberate the free energy required for the membrane fusion event. Indeed, a small-molecule inhibitor of respiratory syncytial virus (RSV) that is postulated to bind to a groove in the HR-A coiled coil (11) and synthetic peptides derived from the HR-B domains of some FMGs are potent inhibitors of viral entry, presumably by competing with the endogenous HR-B sequences for binding to the central HR-A trimer (31, 47, 58, 59). For paramyxoviruses, the fusion (F) protein precursor F0 is cleaved into a larger transmembrane F1 and a smaller extracellular F2 subunit. In addition to the crystal structures of the RSV and simian virus type 5 (SV5) fusion cores, medium- and high-resolution structural information.Residues V459, L457, and L454 are predicted to interact with a hydrophobic groove in the HR-A trimer. 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability. Enveloped viruses, such as retroviruses, paramyxoviruses, orthomyxoviruses, and filoviruses, infect cells through fusion of their lipid envelope with the plasma membrane or intracellular membranes of the target cell (17, 30). For members of these viral families, membrane merger is mediated by homotrimeric type I fusogenic membrane glycoproteins (FMGs), integral membrane proteins displayed on the surfaces of the viral particles (17, 54). All type I FMGs contain an internal hydrophobic domain of approximately 25 amino acids, generally termed the fusion peptide. Proteolytic cleavage at a specific site yields a metastable native FMG that consists of a transmembrane and a membrane-distal subunit. Subsequent activation of the FMG results in insertion of the fusion peptide, which is located in the transmembrane subunit, into the target membrane (21). Depending on the origin of the FMG, activation can be realized at neutral pH, as postulated, for example, for lentiviruses (4, 27) and most paramyxoviruses, including measles virus (MV) (19), or at low-pH conditions in an endosomal compartment of the target cell, as exemplified by influenza virus (54). Insertion of the fusion peptide into the target membrane is then followed by conformational rearrangements of the FMG trimer that bring the fusion peptide and the transmembrane domain, and hence the target and donor membranes, into close proximity (1, 2, 37, 40, 50, 61), ultimately resulting in the formation of a fusion pore. Instrumental in this process are two highly conserved 4-3 heptad repeat (HR) sequences, one of which is located adjacent to the fusion peptide and near the N terminus of the protein (hence termed the HR-N or HR-A domain), while the other is adjacent to the transmembrane domain and near the C terminus (HR-C or HR-B) (17, 29). Activation of the native FMG and insertion of the fusion peptide into the target membrane are thought to be followed by refolding into a transient hairpin intermediate and the formation of a stable six-helix bundle (6-HB) fusion core structure (17, 54). Analysis of this core structure of lentivirus (7) and paramyxovirus (1, 61) FMGs has revealed a central homotrimeric coiled coil formed by HR-A domains that is surrounded by three HR-B helices in an antiparallel fashion (17, 54). In this model, the process of protein refolding and 6-HB formation is thus coupled to membrane fusion (15, 37, 50). The conformational changes may in fact liberate the free energy required for the membrane fusion event. Indeed, a small-molecule inhibitor of respiratory syncytial virus (RSV) that is postulated to bind to a groove in the HR-A coiled coil (11) and synthetic peptides derived from the HR-B domains of some FMGs are potent inhibitors of viral entry, presumably by competing with the endogenous HR-B sequences for binding to the central HR-A trimer (31, 47, 58, 59). For paramyxoviruses, the fusion (F) protein precursor F0 is cleaved into a larger transmembrane F1 and a smaller extracellular F2 subunit. In addition to the crystal structures of the RSV and simian virus type 5 (SV5) fusion cores, medium- and high-resolution structural information for paramyxovirus F proteins comes from a three-dimensional cryoelectron microscopy reconstruction of the Sendai virus F protein (36) and X-ray structures of the Newcastle disease virus (NDV) and 10058-F4 human parainfluenzavirus type 3 (hPIV3) F ectodomains (9, 60). All of.