Functional domains in the retroviral transmembrane protein.

The entry of enveloped viruses into cells requires the fusion of viral and cellular membranes in a process catalyzed by specific viral fusion proteins (41). Fusion is initiated by the binding of the fusion protein itself, or an associated protein, to a specific cellular receptor and involves a series of steps that includes the insertion of a stretch of hydrophobic amino acids (the fusion peptide) into the target cell membrane. The paradigm for viral fusion proteins is the influenza virus hemagglutinin (HA) protein (reviewed in reference 42). The HA1 subunit binds to cell surface sialic acid residues, allowing the virus to be internalized into endosomes by receptor-mediated endocytosis. In the endosome, the low pH triggers conformational changes in HA1 and the associated HA2 subunits, leading to the translocation of the fusion peptide at the N terminus of HA2 toward the target cell membrane (2). An important part of this structural reorganization is the recruitment of part of a heptad repeat sequence in HA2 into a triple-stranded coiled coil (2, 3). Several other viral fusion proteins have been shown to possess features in common with influenza virus HA. Hydrophobic fusion peptides have been identified at the N termini of the transmembrane components of the fusion proteins of the paramyxoviruses and several retroviruses (5, 13), while the avian retroviruses, the filoviruses, and the coronaviruses may also contain fusion peptides in their transmembrane proteins (5, 15). Heptad repeat sequences have been found adjacent to all of these fusion peptides (5, 15), and for human immunodeficiency virus type 1 (HIV-1) (6, 39) and murine leukemia virus (MuLV) (11), these heptad repeats form triple-stranded coiled coils when crystallized. Not all viral fusion proteins are activated by low pH. The paramyxoviruses, coronaviruses, and some retroviruses can fuse at neutral pH and are able to mediate fusion at the cell surface (22, 24, 30, 40). However, conformational changes can be induced in the envelope proteins of HIV-1 (35) and avian sarcoma-leukosis virus (16) by exposure to soluble forms of their receptors, suggesting that structural rearrangements are likely to be a common step in viral fusion, even if triggered by different stimuli. In the MuLVs, a low pH step has been suggested to be a requirement for entry by Moloney MuLV (MoMuLV), because in certain cell lines viral entry is sensitive to lysosomotropic agents (24, 28). However, this sensitivity is cell type dependent, other closely related MuLV strains are not sensitive to such agents, and MuLV fusion proteins cannot be activated by exogenous low-pH treatments (24, 28). Therefore, the reported pH dependence of MoMuLV infection may occur at some step distal to the fusion process. The MuLV envelope protein is initially translated as a precursor protein, Pr85, assembled into oligomers in the endoplasmic reticulum and proteolytically cleaved by a host protease into two subunits, the surface (SU) protein, gp70, and the transmembrane (TM) protein, p15E (8, 9). Crystallographic studies have now provided evidence that the oligomeric form of MoMuLV TM is a trimer (11). At or shortly after the time of virus budding, the TM is further processed by the viral protease to release a 16-amino-acid peptide, the R peptide, from the C terminus of the cytoplasmic tail. Both p15E and the processed form, p12E, coexist in the virion (17, 19). R peptide cleavage of MuLV has profound effects on the fusogenicity of the envelope protein, promoting cell-cell fusion and syncytium formation in NIH 3T3 cells which are not fused by the full-length envelope protein (19, 31, 32). It is likely that the virus has adopted a regulatory mechanism to prevent the premature activation of fusion before the envelope protein is incorporated into a virion, which could be cytopathic to the host cell or interfere with the budding process. The fusogenicity of the envelope proteins of Mason-Pfizer monkey virus (1a) and equine infectious anemia virus (33) is also enhanced by the cleavage of their cytoplasmic tails, and artificial truncations of the cytoplasmic tails of HIV-1, HIV-2 and simian immunodeficiency virus (SIV) have also been shown to enhance fusogenicity (12, 27, 47). We have previously demonstrated that mutant MoMuLV envelope proteins that are defective in either the SU or TM subunits can functionally complement each other when coexpressed, presumably through the formation of hetero-oligomers (45). This observation suggests that the binding signal from one SU monomer can trigger fusion by the associated TM proteins, even when its own TM subunit is defective. We have interpreted these data to indicate that cross talk can occur between monomers of the envelope protein complex. We have now extended those studies to include a panel of fusion-defective mutants that map to distinct predicted features of MoMuLV TM, in order to analyze functional interactions between different regions of the TM protein. In addition, we have compared the ability of R peptide cleavage to enhance fusion in cis to the different TM mutants. In this way, we have been able to identify discrete functional domains within TM, to analyze their interactions, and to suggest how R peptide cleavage may act to modulate the fusogenicity of the envelope protein.