Most class I fusion proteins exist as trimers of dimers composed of a receptor binding and a fusion subunit.In their postfusion forms, the three fusion subunits form trimers of hairpins consisting of a central coiled coil (formed by the N-terminal helices), an intervening sequence, and a region containing the C helix (and flanking strands) that runs antiparallel to and packs in the grooves of the N-terminal coiled coil. For filoviruses and most retroviruses, the intervening sequence includes a "chain reversal region" consisting of a short stretch of hydrophobic residues, a Gly-Gly pair, a CX 6 CC motif, and a bulky hydrophobic residue. Maerz and coworkers (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000) proposed a model for this region of human T-cell leukemia virus type 1 (HTLV-1) Env in which expulsion of the final bulky hydrophobic residue is important for early conformational changes and specific residues in the chain reversal region are important for forming the final, stable trimer of hairpins. Here, we used mutagenesis and pseudovirus entry assays to test this model for the avian retrovirus avian sarcoma/leukosis virus (ASLV) and the filovirus ebolavirus Zaire. Our results are generally consistent with the model proposed for HTLV-1 Env. In addition, we show with ASLV EnvA that the bulky hydrophobic residue following the CX 6 CC motif is required for the step of prehairpin target membrane insertion, whereas other residues are required for the foldback step of fusion. We further found that a His residue that is unique to the chain reversal region of ASLV EnvA controls the pH at which ASLV entry occurs.Class I fusion proteins are trimeric glycoproteins that project from the viral membrane surface. Most harbor the host cell receptor binding and membrane fusion functions within distinct subunits (16,41). Each class I fusion subunit contains a hydrophobic fusion peptide (or fusion loop), two heptad repeats separated by an intervening sequence, a membranespanning sequence, and a cytoplasmic tail. Class I fusion proteins are primed for fusion by one or more proteolytic events that, in most cases, separate the receptor binding and fusion subunits, leaving the fusion peptide/loop at or near the N terminus of the fusion subunit, and most sit on the viral membrane surface in a metastable state in which the receptor binding subunit "clamps" the fusion subunit (21, 42). Receptor binding, decreasing pH (during endocytosis), disulfide exchange, or a combination of these factors triggers release of this clamp (41). Once triggered, the fusion subunit undergoes conformational changes that first extend the fusion peptide/ loop for interaction with the target membrane and then bend the fusion subunit roughly in half, forming a trimer of hairpins that brings the fusion peptides and the membrane-spanning domains, and hence the two membranes they are tethered to, together to create a fusion pore. For class I hairpins, the N-heptad repeats form a trimeric coiled coil, and C-termin...