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. 2001 May;75(9):4268-75.
doi: 10.1128/JVI.75.9.4268-4275.2001.

Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

S L Allison  1 , J Schalich, K Stiasny, C W Mandl, F X Heinz
Affiliations

Mutational evidence for an internal fusion peptide in flavivirus envelope protein E

S L Allison et al. J Virol. 2001 May.

Abstract

The envelope protein E of the flavivirus tick-borne encephalitis (TBE) virus promotes cell entry by inducing fusion of the viral membrane with an intracellular membrane after uptake by endocytosis. This protein differs from other well-studied viral and cellular fusion proteins because of its distinct molecular architecture and apparent lack of involvement of coiled coils in the low-pH-induced structural transitions that lead to fusion. A highly conserved loop (the cd loop), which resides at the distal tip of each subunit and is mostly buried in the subunit interface of the native E homodimer at neutral pH, has been hypothesized to function as an internal fusion peptide at low pH, but this has not yet been shown experimentally. It was predicted by examination of the X-ray crystal structure of the TBE virus E protein (F. A. Rey et al., Nature 375:291-298, 1995) that mutations at a specific residue within this loop (Leu 107) would not cause the native structure to be disrupted. We therefore introduced amino acid substitutions at this position and, using recombinant subviral particles, investigated the effects of these changes on fusion and related properties. Replacement of Leu with hydrophilic amino acids strongly impaired (Thr) or abolished (Asp) fusion activity, whereas a Phe mutant still retained a significant degree of fusion activity. Liposome coflotation experiments showed that the fusion-negative Asp mutant did not form a stable interaction with membranes at low pH, although it was still capable of undergoing the structural rearrangements required for fusion. These data support the hypothesis that the cd loop may be directly involved in interactions with target membranes during fusion.

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Figures

FIG. 1
FIG. 1
(A) Ribbon diagram of the ectodomain portion of the TBE virus E protein dimer (36) viewed from the top (perpendicular to the virion surface), with the individual subunits colored red and blue. The C-terminal portion of the protein, whose structure is not known, extends downward from domain III at each end of the dimer to anchor the protein in the viral membrane. Domains I, II, and III are labeled by roman numerals, and the cd loop at the tip of domain II is colored yellow. The position of Leu 107 is shown by a green ball. (B) Schematic representation of a threefold assembly of E dimers as determined by cryoelectron microscopy and icosahedral reconstruction of RSPs (13). Domains are indicated by roman numerals, and filled yellow circles represent the cd loop of domain II. (C) Zoom of the top view in panel A, showing details of the cd loop and vicinity. Amino acids 100 to 108 (the cd loop) as well as Cys 74 are shown as ball-and-stick representations and colored by atom (green, C; blue, N; red, O; yellow, S). The first N-acetylglucosamine residue of the N-linked glycan, which covers the cd loop but is attached to Asn 154 of the other subunit, is shown in gray. Other amino acids are represented as sticks.
FIG. 2
FIG. 2
Electron micrographs of wild-type and mutant RSPs stained with uranyl acetate.
FIG. 3
FIG. 3
Low-pH-induced fusion of pyrene-labeled RSPs with liposomes. Fusion was measured at 37°C by continuous monitoring of pyrene excimer fluorescence (8), and the extent of fusion was calculated as described in Materials and Methods. At least three replicates were performed, and representative curves are shown. The time zero represents the time of acidification.
FIG. 4
FIG. 4
Antibody binding profiles before and after low-pH treatment. The binding activities of 18 E-protein-specific MAbs with low-pH-treated and untreated RSP-wt (upper panel) and RSP-107D (middle panel) were compared by four-layer ELISA. The blue patterns were obtained with untreated samples, and the red patterns were obtained with samples that had been preincubated at pH 6.0 and back-neutralized. The colors on the x axis of the middle panel represent the structural domains (depicted in the lower panel) to which the antibodies bind (red, domain I; yellow, domain II; blue, domain III). The spheres show the positions of mutations defining individual MAb binding sites that have been mapped by selection of neutralization escape variants of TBE virus (29) or mutations in RSPs that abolish binding of an individual MAb (A1 [this study] or B3 [unpublished data]). The epitopes for the nonneutralizing MAbs B2 and C1 to C6 have not been precisely mapped, but the antigenic domains to which they belong were established previously (29). Note that binding to MAb A1 (arrows) is abolished by the Leu 107-to-Asp mutation.
FIG. 5
FIG. 5
pH dependence of the conformational change in the E protein. RSP-wt and RSP-107D were pretreated at different pHs, and binding activity of MAb A4 with these preparations was measured by four-layer ELISA.
FIG. 6
FIG. 6
Conversion of E homodimers to homotrimers at low pH. RSPs that had been pretreated at the indicated pHs were solubilized with detergent, and sedimentation analysis on continuous sucrose gradients was used to assess the oligomeric state of the E protein. The positions of the E dimer and trimer peaks are indicated.
FIG. 7
FIG. 7
Liposome coflotation assay. RSPs were incubated with liposomes at the indicated pHs and analyzed by sucrose step gradient centrifugation. The positions of the sucrose layers are indicated below each graph, and the 20% sucrose layer is shaded to indicate the position where the samples were initially applied before centrifugation.
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References

    1. Allison S L, Mandl C W, Kunz C, Heinz F X. Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR. Virus Genes. 1994;8:187–198. - PubMed
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