The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the aminoterminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the aminoterminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.Cell entry by retroviruses is mediated by the virally encoded Env glycoprotein on the virus surface. The Env protein of Moloney murine leukemia virus (MoMLV), the best-characterized mammalian type C retrovirus, is synthesized as a single 85-kDa precursor that includes an amino-terminal signal sequence that directs its insertion into the lumen of the endoplasmic reticulum (ER) (61). During transport to the cell surface, the Env precursor undergoes a series of posttranslational modifications, including glycosylation, oligomerization, disulfide bond formation and cleavage into surface (SU) and transmembrane (TM) subunits (19,46,47,48). At the cell surface, the Env trimer is incorporated into the membrane of the budding virion, where it is further modified by the virally encoded protease, which removes the carboxy-terminal 16 amino acids (the R-peptide) of TM to yield the entry-competent glycoprotein (22, 50, 51, 52).The entry process is initiated by the specific interaction of the Env protein with the receptor protein MCAT-1, which is located on the surface of a susceptible cell (1). Receptorbound virion particles are then internalized, probably by clathrin-independent endocytosis (38). By analogy with the influenza hemagglutinin (HA) fusion protein, the reduced pH within the endosome mediates a conformational change in the Moloney Env protein that exposes the fusion peptide at the amino terminus of TM (55). Fusion, initiated by the insertion of the fusion peptide into the endosomal membrane, permits ...