Role of the cytoplasmic tail of ecotropic moloney murine leukemia virus Env protein in fusion pore formation.
Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.
Melikyan
G B
GB
Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA.
Markosyan
R M
RM
Brener
S A
SA
Rozenberg
Y
Y
Cohen
F S
FS
D016475:3T3 Cells; D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism*; D015686:Gene Products, env / Q000502:physiology*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000378:metabolism*
D015686:Gene Products, env