A Rotavirus Spike Protein Conformational Intermediate Binds Lipid Bilayers

Abstract

During rotavirus entry, a virion penetrates a host cell membrane, sheds its outer capsid proteins, and releases a transcriptionally active subviral particle into the cytoplasm. VP5*, the rotavirus protein believed to interact with the membrane bilayer, is a tryptic cleavage product of the outer capsid spike protein, VP4. When a rotavirus particle uncoats, VP5* folds back, in a rearrangement that resembles the fusogenic conformational changes in enveloped-virus fusion proteins. We present direct experimental evidence that this rearrangement leads to membrane binding. VP5* does not associate with liposomes when mounted as part of the trypsinprimed spikes on intact virions, nor does it do so after it has folded back into a stably trimeric, low-energy state. But it does bind liposomes when they are added to virions before uncoating, and VP5* rearrangement is then triggered by addition of EDTA. The presence of liposomes during the rearrangement enhances the otherwise inefficient VP5* conformational change. A VP5* fragment, VP5CT, produced from monomeric recombinant VP4 by successive treatments with chymotrypsin and trypsin, also binds liposomes only when the proteolysis proceeds in their presence. A monoclonal antibody that neutralizes infectivity by blocking a postattachment entry event also blocks VP5* liposome association. We propose that VP5* binds lipid bilayers in an intermediate conformational state, analogous to the extended intermediate conformation of enveloped-virus fusion proteins.

Cross-Linking of Rotavirus Outer Capsid Protein VP7 by Antibodies or Disulfides Inhibits Viral Entry

“…The slight (43.5%) reduction observed at the highest concentration could be due to a trace of remaining intact IgG, nonspecific interference with viral uptake when particles are fully decorated with Fab, or steric interference with VP4 conformational changes necessary for viral entry (14,57,61). Bridging two RV3:4 Fabs with a Fab-directed secondary antibody restored neutralization.…”

Effect of Mutations in VP5* Hydrophobic Loops on Rotavirus Cell Entry

“…WT , as assayed by cosedimentation on a sucrose gradient (29). When digested in the presence of liposomes, each of the mutant VP4s produced a VP5CT-like molecule that was recognized by the trimer-specific MAb 4D8 (32) in immunoblots of unheated samples from a sucrose gradient separation of the digestion products (Fig.…”

“…Liposome association reactions were performed as described previously (29). In brief, phosphatidylcholine (PC) (from chicken eggs; Avanti Polar Lipids) liposomes were formed in HN (20 mM HEPES, pH 7.3, 140 mM NaCl) at a concentration of 10 mg/ml.…”

“…Structural analyses of various VP4 domains, of VP7, and of DLPs and TLPs (2,6,10,11,18,31,33), together with biochemical studies of VP7, VP4, and VP4 fragments (8,9,28,29,32), suggest the model illustrated in Fig. 1.…”

“…1) and for its interaction with synthetic membranes (29). Digestion of recombinant VP4 with chymotrypsin and trypsin in the presence of liposomes leads to membrane association of the resulting VP5CT, but preformed VP5CT does not associate with liposomes added after cleavage and trimerization are complete.…”