Prefusion Rearrangements Resulting in Fusion Peptide Exposure in Semliki Forest Virus

Hammar, Lena Marmstål; Markarian, Sevak; Haag, Lars; Lankinen, Hilkka; Salmi, A.; Cheng, R. Holland

doi:10.1074/jbc.m206015200

Cited by 28 publications

(31 citation statements)

References 77 publications

(50 reference statements)

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“…Virus Purification by Tartrate Gradient Centrifugation and Quality Control-The SFV4 was propagated and purified, essentially as described previously (29,39). The SFV SQL was activated for infection by chymotrypsin treatment but otherwise handled in the same way as the wild type virus.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we tested the exposure of the fusion loop relative pH as the capacity to bind mAb E1f, an antibody that recognizes the fusion loop sequence (39). Thus, coated in an ELISA plate at the same protein load, the particles were exposed to mAb E1f at different pH, and the bound antibody, reflecting the accessible sequence, was measured.…”

Section: The Ph Profile Of Fusion Loopmentioning

confidence: 99%

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The Dynamic Envelope of a Fusion Class II Virus

Haag²,

Sjöberg

et al. 2008

Journal of Biological Chemistry

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In alphaviruses, here represented by Semliki Forest virus, infection requires an acid-responsive spike configuration to facilitate membrane fusion. The creation of this relies on the chaperon function of glycoprotein E2 precursor (p62) and its maturation cleavage into the small external E3 and the membrane-anchored E2 glycoproteins. To reveal how the E3 domain of p62 exerts its control of spike functions, we determine the structure of a p62 cleavage-impaired mutant virus particle (SQL) by electron cryomicroscopy. A comparison with the earlier solved wild type virus structure reveals that the E3 domain of p62 SQL forms a bulky side protrusion in the spike head region. This establishes a gripper over part of domain II of the fusion protein, with a cotter-like connection downward to a hydrophobic cluster in its central ␤-sheet. This finding reevaluates the role of the precursor from being only a provider of a shield over the fusion loop to a structural playmate in formation of the fusogenic architecture.When an enveloped virus infects its target cell, the mechanism usually involves a step with hairpin refolding of the viral fusion protein to promote merging of virus and target membranes. Opposite to the class I fusion proteins, which are trimers from the start and in which the fusion-related refolding involves formation and backfolding of ␣-helical bundles, the class II fusion proteins elaborate on homotrimer formation to mediate membrane fusion with target membrane, as discussed by Kielian (1, 2) and others (3-5). In the alphaviruses, here represented by Semliki Forest virus (SFV), 3 the fusion proteins are of class II and essentially lack helical motifs. Homotrimers of the fusion protein E1, formed in relation to the membrane fusion process, are stable associations (6 -13). The strong interaction would be the driving force for completion of fusion, after low pH and membrane contact trigger. However, it would be suicidal for virus transmission if the fusion protein were allowed to create such a configuration prematurely. The SFV assembly pathway handles this problem by providing a chaperon protein, the precursor of glycoprotein E2, in SFV named p62. The p62 precedes the E1 in the proprecursor sequence (p62-6K-E1; see Scheme 1) and waits in the ER to form dimers with the nascent E1, thereby allowing transport to the Golgi compartment and forestalling E1 self-aggregation (10, 15). The E2 itself, if translocated into ER by a cleavable signal sequence, is not sufficient for the purpose. This was shown with an E3 deletion mutant, where the N-terminal E3 domain of the precursor was exchanged for a cleavable artificial signal sequence to preserve the membrane topology of authentic E2. In this E3 deletion mutant, expressed via a recombinant vaccinia virus, the heterodimerization of the spike proteins was abolished, and the E1 was completely retained in the ER (14). In the early Golgi compartment, the p62-E1 heterodimers may form trimers of dimers (16) that in the trans-Golgi undergo furin-dependent maturation cleavage (9,...

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Section: Methodsmentioning

confidence: 99%

Section: The Ph Profile Of Fusion Loopmentioning

confidence: 99%

The Dynamic Envelope of a Fusion Class II Virus

Haag²,

Sjöberg

et al. 2008

Journal of Biological Chemistry

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“…Neutralizing antibodies directed against these fusion regions have been well described in other virus systems, including closely related flaviviruses (63), more distantly related alphaviruses (64), and unrelated orthomyxoviruses (65). Our results are consistent with those of a recent study that identified two other broadly neutralizing hMAbs from a single patient that target DENV DI/II and whose binding to WNV DI/II was ablated when residues in the fusion loop were altered, suggesting that these antibodies may also bind to the DENV fusion loop (32).…”

Section: Figmentioning

confidence: 99%

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

Costin

Зайцева

Kahle

et al. 2013

J Virol

117

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There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.

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“…The epitope for MAb E1f was previously mapped to residues 85 to 95 of the fusion peptide (15), and our studies have shown that this epitope is masked when E1 ‫ء‬ inserts into cholesterol-containing target membranes at low pH (1 COS-7 cell expression vectors and transfection. For expression in COS-7 cells, the vectors pL2-SFV-88 and pL2-SFV-⌬83-92 were used, encoding the wild-type structural proteins and the structural proteins with a deletion of residues 83 to 92 of E1, respectively.…”

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confidence: 99%

Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus

Gibbons

Ahn

Liao

et al. 2004

J Virol

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A prevailing model for virus membrane fusion proteins has been that the hydrophobic fusion peptide is hidden in the prefusion conformation, becomes exposed once the fusion reaction is triggered, and then either inserts into target membranes or is rapidly inactivated. This model is in general agreement with the structure and mechanism of class I fusion proteins, such as the influenza virus hemagglutinin. We here describe studies of the class II fusion protein E1 from the alphavirus Semliki Forest virus (SFV). SFV fusion is triggered by low pH, which releases E1 from its heterodimeric interaction with the E2 protein and induces the formation of a stable E1 homotrimer. The exposure and target membrane interaction of the E1 fusion peptide (residues 83 to 100) were followed using a monoclonal antibody (MAb E1f) mapping to E1 residues 85 to 95. In agreement with the known structure of SFV and other alphaviruses, the fusion peptide was shielded in native SFV particles and exposed when E1-E2 dimer dissociation was triggered by acidic pH. In contrast, the fusion peptide on purified E1 ectodomains (E1 ‫ء‬ ) was fully accessible at neutral pH. Functional assays showed that MAb E1f binding at neutral pH prevented subsequent low-pH-triggered E1‫ء‬ interaction with target membranes and trimerization. E1 ‫ء‬ was not inactivated by low pH when treated either in the absence of target membranes or in the presence of fusion-inactive cholesterol-deficient liposomes. Thus, the membrane insertion of the E1 fusion peptide is regulated by additional low-pH-dependent steps after exposure, perhaps involving an E1-cholesterol interaction.Enveloped viruses catalyze a membrane fusion reaction between the virus and cellular membranes in order to infect the cell. This fusion event is mediated by a viral membrane glycoprotein through its dual interaction with the virus membrane and the cellular target membrane. The region of the virus fusion protein that inserts directly into the target membrane is termed the fusion peptide. Although they have not been rigorously identified in many viruses, in general fusion peptides are N-terminal or internal regions of ϳ16 to 36 residues that are relatively hydrophobic in nature and are highly conserved within virus families (42). Since the interaction of the fusion peptide with the target membrane is critical to the fusion reaction, enveloped viruses must appropriately regulate their fusion proteins such that the fusion peptide-membrane interaction occurs only at the proper time and place.The mechanism and regulation of viral fusion proteins are best understood for the influenza virus hemagglutinin (HA) protein, the prototype of the class I viral fusion proteins (reviewed in references 10 and 33). Class I fusion proteins are native trimers whose fusion can be triggered by endocytic low pH, as in the case of HA, or by receptor and/or coreceptor interactions. Fusion involves the refolding of the fusion protein to a six-helix bundle with a central coiled-coil and the fusion peptide and transmembrane domain at ...

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Prefusion Rearrangements Resulting in Fusion Peptide Exposure in Semliki Forest Virus

Cited by 28 publications

References 77 publications

The Dynamic Envelope of a Fusion Class II Virus

The Dynamic Envelope of a Fusion Class II Virus

Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

Multistep Regulation of Membrane Insertion of the Fusion Peptide of Semliki Forest Virus

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