Site-directed antibodies against the stem region reveal low pH-induced conformational changes of the Semliki Forest virus fusion protein.
The E1 envelope protein of the alphavirus Semliki Forest virus (SFV) is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. Like other class I and class II fusion proteins, during fusion E1 inserts into the target membrane and rearranges to form a trimeric hairpin structure. The postfusion structures of the alphavirus and flavivirus fusion proteins suggest that the "stem" region connecting the fusion protein domain III to the transmembrane domain interacts along the trimer core during the low pH-induced conformational change. However, the location of the E1 stem in the SFV particle and its rearrangement and functional importance during fusion are not known. We developed site-directed polyclonal antibodies to the N- or C-terminal regions of the SFV E1 stem and used them to study the stem during fusion. The E1 stem was hidden on neutral pH virus but became accessible after low pH-triggered dissociation of the E2/E1 heterodimer. The stem packed onto the trimer core in the postfusion conformation and became inaccessible to antibody binding. Generation of the E1 homotrimer on fusion-incompetent membranes identified an intermediate conformation in which domain III had folded back but stem packing was incomplete. Our data suggest that E1 hairpin formation occurs by the sequential packing of domain III and the stem onto the trimer core and indicate a tight correlation between stem packing and membrane merger.
Liao
Maofu
M
Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Kielian
Margaret
M
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D012672:Semliki forest virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D000914:Antibodies, Viral; D014760:Viral Fusion Proteins