Acid-Induced Changes in Thermal Stability and Fusion Activity of Influenza Hemagglutinin

Remeta, David P.; Krumbiegel, M.; Minetti, Conceição A.S.A.; Puri, Anu; Ginsburg, Ann; Blumenthal, Robert

doi:10.1021/bi015614a

Cited by 35 publications

(38 citation statements)

References 51 publications

(97 reference statements)

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“…There are two components of the thermal transition: one around 65°C and the other at about 85°C. The lower temperature transition is similar to that seen with the intact protein (24,25). The transitions are partially reversible on cooling.…”

Section: Resultssupporting

confidence: 66%

“…For example, a small difference in the pH dependence of hemolysis between an intact influenza virus and the isolated hemagglutinin protein has been observed (34). The detailed study of the pH dependence of fusion by Remeta et al (24) shows that at 35°C the pH required for half-maximal activity for lipid mixing with erythrocyte ghosts was 5.50. The pH required for half-maximal promotion of heterokaryon formation with cells expressing the influenza virus X-31 hemagglutinin protein is 5.2 (35), whereas the pH required for half-maximal hemolytic activity is 5.75 (36).…”

Section: Discussionmentioning

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: Resultssupporting

confidence: 66%

Section: Discussionmentioning

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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“…The molecular basis for pH-induced activation of envelope glycoprotein-mediated membrane fusion is largely undefined. Despite detailed structural knowledge of membrane fusion in the best-studied pH-dependent envelope glycoprotein, influenza virus hemagglutinin (HA), key elements of the activation process remain to be completely elucidated (9,43,50). Studies of tert-butyl hydroquinone HA fusion inhibitors have demonstrated the metastable nature of the prefusion HA complex and the ability of small molecules to stabilize against acidic pHs (2,27).…”

Section: Discussionmentioning

confidence: 99%

pH-Induced Activation of Arenavirus Membrane Fusion Is Antagonized by Small-Molecule Inhibitors

York

Dai

Amberg

et al. 2008

J Virol

159

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The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPCmediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.

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“…Finally, pH-induced conformational changes also have been shown to confer the capacity to integrate into membranes. Examples are seen in clathrin (Maezawa et al, 1989), Human rhinovirus type 2 (Brabec et al, 2003), and Influenza virus hemagglutinin (Remeta et al, 2002).…”

Section: Membrane Association Of Gfp:mpmentioning

confidence: 99%

Involvement of the Secretory Pathway and the Cytoskeleton in Intracellular Targeting and Tubule Assembly of Grapevine fanleaf virus Movement Protein in Tobacco BY-2 Cells

et al. 2003

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Acid-Induced Changes in Thermal Stability and Fusion Activity of Influenza Hemagglutinin

Cited by 35 publications

References 51 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

pH-Induced Activation of Arenavirus Membrane Fusion Is Antagonized by Small-Molecule Inhibitors

Involvement of the Secretory Pathway and the Cytoskeleton in Intracellular Targeting and Tubule Assembly of Grapevine fanleaf virus Movement Protein in Tobacco BY-2 Cells

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