Completion of trimeric hairpin formation of influenza virus hemagglutinin promotes fusion pore opening and enlargement

Borrego-Diaz, Emma; Peeples, Mark E.; Markosyan, Ruben M.; Melikyan, Gregory B.; Cohen, Fredric S.

doi:10.1016/j.virol.2003.07.006

Cited by 36 publications

(46 citation statements)

References 48 publications

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“…Note that many of the curvature-generating proteins involved in membrane remodeling lack transmembrane domains (66). The finding that HA2* does not produce fusion pores large enough to allow passage of 10-kDa fluorescent dextran (Stokes radius of 2.4 nm) and does not produce syncytia substantiates the hypothesis that the transmembrane domain (and perhaps its interactions with the fusion peptide) as well as the cytoplasmic domain of HA can be especially important for later fusion stages including fusion pore expansion (26,57,67,68).…”

Section: Discussionmentioning

confidence: 75%

“…4, A and B, and 5A). A single amino acid substitution at position 173 (I173E), detrimental for HA-mediated fusion (26), significantly inhibited the fusogenic activity of HA2*. Similarly, a substitution of glutamic acid for the glycine in the N terminus (G1E) of HA2 that is known to inhibit HA-mediated fusion (22) resulted in an almost complete loss of the fusogenic activity of the HA2*.…”

Section: Resultsmentioning

confidence: 99%

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The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

Kim

Epand

Leikina

et al. 2011

Journal of Biological Chemistry

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One of the best characterized fusion proteins, the influenza virus hemagglutinin (HA), mediates fusion between the viral envelope and the endosomal membrane during viral entry into the cell. In the initial conformation of HA, its fusogenic subunit, the transmembrane protein HA2, is locked in a metastable conformation by the receptor-binding HA1 subunit of HA. Acidification in the endosome triggers HA2 refolding toward the final lowest energy conformation. Is the fusion process driven by this final conformation or, as often suggested, by the energy released by protein restructuring? Here we explored structural properties as well as the fusogenic activity of the full sized trimeric HA2(1-185) (here called HA2*) that presents the final conformation of the HA2 ectodomain. We found HA2* to mediate fusion between lipid bilayers and between biological membranes in a low pH-dependent manner. Two mutations known to inhibit HA-mediated fusion strongly inhibited the fusogenic activity of HA2*. At surface densities similar to those of HA in the influenza virus particle, HA2* formed small fusion pores but did not expand them. Our results confirm that the HA1 subunit responsible for receptor binding as well as the transmembrane and cytosolic domains of HA2 is not required for fusion pore opening and substantiate the hypothesis that the final form of HA2 is more important for fusion than the conformational change that generates this form.Fusion mediated by the influenza virus hemagglutinin (HA) protein is often considered as a prototype of biological fusion reactions. This fusion process is utilized by the virus to deliver its RNA into the host cell by merging the viral envelope with the membrane of an acidified endosome of the host cell. Each monomer of homotrimeric HA consists of two disulfide-linked subunits: HA1, responsible for receptor binding, and HA2. In the native neutral pH conformation of the HA protein, the HA1 subunit confines the HA2 subunit in a metastable state that has been termed the "spring-loaded" state (1-3). However, evidence from calorimetric studies indicates that the compact folded structure of the influenza hemagglutinin protein is not a kinetically trapped metastable high energy form (4, 5). In the neutral pH structure of intact HA, the functionally important N-terminal amphiphilic region of HA2, referred to as the fusion peptide, is hidden within the HA molecule. The low pH-triggered restructuring of HA unlocks HA2 and allows refolding of the HA2 subunit toward its final, low energy conformation, which consists of a hairpin structure with the fusion peptide and the transmembrane domain at the same end of the rigid rod. Although it is commonly assumed that the energy released by this restructuring drives rearrangements of membrane bilayers (6 -8), one may suggest an alternative hypothesis, i.e. that the final conformation itself is fusogenic.In our earlier work, to test the fusogenic properties of the final conformation of the HA2 subunit in the absence of

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Section: Discussionmentioning

confidence: 75%

Section: Resultsmentioning

confidence: 99%

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

Kim

Epand

Leikina

et al. 2011

Journal of Biological Chemistry

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“…However, it was subsequently shown that for HIV Env, the formation of those 6HBs that participate in fusion occurs late in the process, and bundle formation is completed only after a pore has formed (28). In contrast to HIV Env, 6HBs may form prior to pore formation for Env of avian sarcoma and leukosis virus (27) and for HA of influenza virus (5,39). For HIV Env, the sequence of amino acids intervening between the fusion peptide and HR2 is relatively short, whereas for avian sarcoma and leukosis virus Env and influenza HA, it is much longer.…”

Section: Discussionmentioning

confidence: 99%

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Markosyan

Leung

Cohen

2009

J Virol

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Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.All class I viral fusion proteins are composed of three identical monomers that together fold into a six-helix bundle (6HB) in the protein's final, postfusion state (33, 50). Each monomer of human immunodeficiency virus (HIV) Env (which is a class I fusion protein) consists of two subunits, gp120 and gp41. The three gp41 subunits of each Env trimer form the 6HB. The crystallographic structure of gp41 has been determined for its final state; its 6HB reveals that the three N-terminal segments of gp41-each segment referred to as heptad repeat 1 (HR1)-have folded into a central triple-stranded coiled coil of ␣-helices, and the three C-terminal segments (HR2) have packed, antiparallel, as ␣-helices into the three grooves of the coiled coil. The 6HB of HIV Env is thermally stable (43) and can confidently be considered a final structure.The crystallographic structures for both the initial and final states have been obtained for several other class I viral fusion proteins (2,7,22,26,43,54). They reveal that the protein must undergo large-scale conformational changes in transiting from their initial state to final 6HB state, including changes in secondary structure. Although the initial structure of gp41 has not been determined, it is known that grooves are not exposed in its initial conformation; they become exposed as part of the gp41 conformational changes during fusion (13). The configurations of Env during which grooves are exposed are referred to as prebundles. It is assumed that gp41 undergoes large-scale reconfigurations during the fusion process.It is certain that formation of the 6HB is a central event in infection; 6HBs form in all class I proteins; peptides that prevent formation of the bundle b...

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“…Subsequently, HRB binds into the grooves between adjacent HRA monomers in an antiparallel orientation, forming a hairpin structure that brings the FP and TM domains into juxtaposition (12, 64). Peptides derived from the HR regions inhibit viral entry by binding specifically to their complementary regions in the vFGP (8, 54, 58), thereby preventing formation of the hairpin structure which couples protein refolding directly to membrane fusion (5,38,42,57). While most class I vFGPs have short linker regions between their HR regions, the paramyxovirus F protein has a two-domain insertion of approximately 250 residues (10) which may serve as a molecular scaffold to help control the release and refolding of the FP and HR regions.…”

mentioning

confidence: 99%

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

Russell

Jardetzky

Lamb

2004

J Virol

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Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation.The class I viral fusion-mediating glycoproteins (vFGPs) of retroviruses, lentiviruses, filoviruses, coronaviruses, orthomyxoviruses, and paramyxoviruses have evolved similar domain architectures to cause the coalescence of viral and host cell membranes. These vFGPs are synthesized as type I integral membrane proteins which are folded into homotrimers, posttranslationally modified by the addition of carbohydrate chains, and then in most cases cleaved by a protease into a metastable complex of membrane-distal and membrane-anchored subunits. For example, the paramyxovirus precursor fusion protein (F 0 ) is cleaved into membrane-distal F 2 and membrane-anchored F 1 subunits (Fig. 1) which have an integrated and not separate domain structure in the atomic structure of F (10). The membrane-distal subunits from many class I vFGPs have insertions of receptor-binding domains (12, 64); however, the paramyxoviruses have evolved separate receptor-binding proteins to regulate fusion activation (33). The membrane-proximal subunits contain two hydrophobic domains, the fusion peptide (FP) and the transmembrane (TM) domain. The FP is located at or near the new N terminus of the membraneproximal subunit and inserts into the target cell membrane after being triggered by low pH and/or receptor binding (12,64). The TM domain serves as an anchor for the ectodomain to the viral membrane and is required for fusion pore formation (1, 39). Adjacen...

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Completion of trimeric hairpin formation of influenza virus hemagglutinin promotes fusion pore opening and enlargement

Cited by 36 publications

References 48 publications

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

The Final Conformation of the Complete Ectodomain of the HA2 Subunit of Influenza Hemagglutinin Can by Itself Drive Low pH-dependent Fusion

The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

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