A study of low pH-induced refolding of Env of avian sarcoma and leukosis virus into a six-helix bundle.
The fusion protein of avian sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of avian sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37 degrees C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.
Markosyan
R M
RM
Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois 60612, USA.
Bates
P
P
Cohen
F S
FS
Melikyan
G B
GB
D016475:3T3 Cells; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D015686:Gene Products, env / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion / Q000187:drug effects* / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D010455:Peptides / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects
D015686:Gene Products, env; D010455:Peptides