Furin cleavage potentiates the membrane fusion-controlling intersubunit disulfide bond isomerization activity of leukemia virus Env.
The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.
Sjöberg
Mathilda
M
Department of Biosciences at Novum, Karolinska Institute, S-141 57 Huddinge, Sweden.
Wallin
Michael
M
Lindqvist
Birgitta
B
Garoff
Henrik
H
D004220:Disulfides / Q000378:metabolism*; D045683:Furin / Q000378:metabolism*; D015752:Genes, env / Q000502:physiology; D007536:Isomerism; D009052:Leukemia Virus, Murine / Q000502:physiology*; D020033:Protein Isoforms; D011991:Receptors, Virus / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000378:metabolism
D004220:Disulfides; D020033:Protein Isoforms; D011991:Receptors, Virus; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins; D045683:Furin