scholarly journals A Point Mutation in the Transmembrane Domain of the Hemagglutinin of Influenza Virus Stabilizes a Hemifusion Intermediate That Can Transit to Fusion

2000 ◽  
Vol 11 (11) ◽  
pp. 3765-3775 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Ruben M. Markosyan ◽  
Michael G. Roth ◽  
Fredric S. Cohen
Keyword(s):  
Influenza Virus ◽  
Point Mutation ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Proteinase K ◽  
Wild Type ◽  
Decreasing Ph ◽  
Lower Temperature ◽  
Increasing Temperature ◽  
Fusion Pores

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23°C rather than 37°C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37°C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4°C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

scholarly journals Inner but Not Outer Membrane Leaflets Control the Transition from Glycosylphosphatidylinositol-anchored Influenza Hemagglutinin-induced Hemifusion to Full Fusion

1997 ◽  
Vol 136 (5) ◽  
pp. 995-1005 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sofya A. Brener ◽  
Dong C. Ok ◽  
Fredric S. Cohen
Keyword(s):  
Outer Membrane ◽  
Negative Curvature ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Positive Curvature ◽  
Fusion Pore ◽  
Spontaneous Curvature ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
Fusion Pores

Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that express the HA-ectodomain anchored to membranes by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane leaflets to determine the contribution of each leaflet in the transition from hemifusion to fusion. When inserted into outer leaflets, amphipaths did not promote the transition, independent of whether the agent induces monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This suggests that fusion is completed when a lipidic fusion pore with net positive curvature is formed by the inner leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound to cells expressing wild-type HA so that lipid but not aqueous dye spread was observed. While this is the same pattern of dye spread as in stable hemifusion, for this “stunted” fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation and enlargement of fusion pores induced by HA. We propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation by conferring positive spontaneous curvature to leaflets of the hemifusion diaphragm.

scholarly journals Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

1999 ◽  
Vol 10 (6) ◽  
pp. 1821-1836 ◽  
Author(s):  
Grigory B. Melikyan ◽  
Sasa Lin ◽  
Michael G. Roth ◽  
Fredric S. Cohen
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Membrane Fusion ◽  
Integral Membrane Protein ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Tm Domain ◽  
Fusion Pores ◽  
Sequence Requirements

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.

scholarly journals The Lipid-anchored Ectodomain of Influenza Virus Hemagglutinin (GPI-HA) Is Capable of Inducing Nonenlarging Fusion Pores

2000 ◽  
Vol 11 (4) ◽  
pp. 1143-1152 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Influenza Virus ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Initial Membrane ◽  
Optimal Fusion ◽  
Pass Through

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.

Familial essential thrombocythemia associated with a dominant-positive activating mutation of the c-MPL gene, which encodes for the receptor for thrombopoietin

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4198-4200 ◽  
Author(s):  
Jianmin Ding ◽  
Hirokazu Komatsu ◽  
Atsushi Wakita ◽  
Miyuki Kato-Uranishi ◽  
Masato Ito ◽  
...  
Keyword(s):  
Point Mutation ◽  
Essential Thrombocythemia ◽  
Intracellular Signaling ◽  
Autosomal Dominant ◽  
Transmembrane Domain ◽  
The Other ◽  
Unique Point ◽  
Wild Type ◽  
Activating Mutation ◽  
Survival Capacity

Abstract One Japanese pedigree of familial essential thrombocythemia (FET) inherited in an autosomal-dominant manner is presented. A unique point mutation, serine 505 to asparagine 505 (Ser505Asn), was identified in the transmembrane domain of the c-MPL gene in all of the 8 members with thrombocythemia, but in none of the other 8 unaffected members in this FET family. The Ba/F3 cells expressing the mutant Asn505 acquired interleukin 3 (IL-3)-independent survival capacity, whereas those expressing wild-type Ser505 did not. The autonomous phosphorylation of Mek1/2 and Stat5b was observed in the mutant Ba/F3 cells in the absence of IL-3. The former was also found in platelets derived from the affected individual in the absence of thrombopoietin. These results show that the Asn505 is an activating mutation with respect to the intracellular signaling and survival of the cells. This is the first report of FET deriving from a dominant-positive activating mutation of the c-MPL gene. (Blood. 2004;103: 4198-4200)

scholarly journals Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

2000 ◽  
Vol 74 (16) ◽  
pp. 7529-7537 ◽  
Author(s):  
Christine Kozerski ◽  
Evgeni Ponimaskin ◽  
Britta Schroth-Diez ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Expression System ◽  
Fusion Pore ◽  
Target Cells ◽  
Fusion Activity ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Pore Enlargement

ABSTRACT The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

scholarly journals High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction

2015 ◽  
Vol 112 (46) ◽  
pp. 14260-14265 ◽  
Author(s):  
Jessica L. Thomaston ◽  
Mercedes Alfonso-Prieto ◽  
Rahel A. Woldeyes ◽  
James S. Fraser ◽  
Michael L. Klein ◽  
...  
Keyword(s):  
High Resolution ◽  
Influenza A Virus ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Proton Channel ◽  
The Matrix ◽  
Crystallographic Structures ◽  
Decreasing Ph ◽  
Dynamics Simulations ◽  
Increasing Temperature

The matrix 2 (M2) protein from influenza A virus is a proton channel that uses His37 as a selectivity filter. Here we report high-resolution (1.10 Å) cryogenic crystallographic structures of the transmembrane domain of M2 at low and high pH. These structures reveal that waters within the pore form hydrogen-bonded networks or “water wires” spanning 17 Å from the channel entrance to His37. Pore-lining carbonyl groups are well situated to stabilize hydronium via second-shell interactions involving bridging water molecules. In addition, room temperature crystallographic structures indicate that water becomes increasingly fluid with increasing temperature and decreasing pH, despite the higher electrostatic field. Complementary molecular dynamics simulations reveal a collective switch of hydrogen bond orientations that can contribute to the directionality of proton flux as His37 is dynamically protonated and deprotonated in the conduction cycle.

scholarly journals Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

2005 ◽  
Vol 79 (10) ◽  
pp. 6449-6458 ◽  
Author(s):  
Ralf Wagner ◽  
Astrid Herwig ◽  
Nahid Azzouz ◽  
Hans Dieter Klenk
Keyword(s):  
Influenza Virus ◽  
Wild Type Virus ◽  
Fusion Pore ◽  
Cysteine Residues ◽  
Virus Infectivity ◽  
Wild Type ◽  
Protein Patterns ◽  
Virus Growth ◽  
Influenza Virus Hemagglutinin ◽  
Fusion Pores

ABSTRACT Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity.

scholarly journals Fatty Acids on the A/USSR/77 Influenza Virus Hemagglutinin Facilitate the Transition from Hemifusion to Fusion Pore Formation

2002 ◽  
Vol 76 (9) ◽  
pp. 4603-4611 ◽  
Author(s):  
Tatsuya Sakai ◽  
Reiko Ohuchi ◽  
Masanobu Ohuchi
Keyword(s):  
Influenza Virus ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Fusion Reaction ◽  
Biological Significance ◽  
Site Directed Mutagenesis ◽  
Fusion Pore ◽  
Influenza Virus Hemagglutinin ◽  
Calcein Leakage ◽  
Palmitic Acids

ABSTRACT Influenza virus hemagglutinin (HA) has three highly conserved acylation sites close to the carboxyl terminus of the HA2 subunit, one in the transmembrane domain and two in the cytoplasmic domain. Each site is modified by palmitic acid through a thioester linkage to cysteine. To elucidate the biological significance of HA acylation, the acylation sites of HA of influenza virus strain A/USSR/77 (H1N1) were changed by site-directed mutagenesis, and the membrane fusion activity of mutant HAs lacking the acylation site(s) was examined quantitatively using transfer assays of lipid (R18) and aqueous (calcein) dyes. Lipid mixing, so-called hemifusion, activity was not affected by deacylation, whereas transfer of aqueous dye, so-called fusion pore formation, was dramatically restricted. When the fusion reaction was induced by a lower pH than the optimal one, calcein transfer with the mutant HAs was improved, but simultaneously a considerable calcein leakage into the medium was observed. From these results, we conclude that the palmitic acids on the H1 subtype HA facilitate the transition from hemifusion to fusion pore formation.

scholarly journals Influenza Virus Hemagglutinin (H3 Subtype) Requires Palmitoylation of Its Cytoplasmic Tail for Assembly: M1 Proteins of Two Subtypes Differ in Their Ability To Support Assembly

2005 ◽  
Vol 79 (21) ◽  
pp. 13673-13684 ◽  
Author(s):  
Benjamin J. Chen ◽  
Makoto Takeda ◽  
Robert A. Lamb
Keyword(s):  
Influenza Virus ◽  
Virus Replication ◽  
Influenza A ◽  
Transmembrane Domain ◽  
Mdck Cells ◽  
Virus Assembly ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Ha Gene

ABSTRACT The influenza A virus hemagglutinin (HA) transmembrane domain boundary region and the cytoplasmic tail contain three cysteines (residues 555, 562, and 565 for the H3 HA subtype) that are highly conserved among the 16 HA subtypes and which are each modified by the covalent addition of palmitic acid. Previous analysis of the role of these conserved cysteine residues led to differing data, suggesting either no role for HA palmitoylation or an important role for HA palmitoylation. To reexamine the role of these residues in the influenza virus life cycle, a series of cysteine-to-serine mutations were introduced into the HA gene of influenza virus A/Udorn/72 (Ud) (H3N2) by using a highly efficient reverse genetics system. Mutant viruses containing HA-C562S and HA-C565S mutations had reduced growth and failed to form plaques in MDCK cells but formed wild-type-like plaques in an MDCK cell line expressing wild-type HA. In cell-cell fusion assays, nonpalmitoylated H3 HA, in both cDNA-transfected and virus-infected cells, was fully competent for HA-mediated membrane fusion. When the HA cytoplasmic tail cysteine mutants were examined for lipid raft association, using as the criterion Triton X-100 insolubility, loss of raft association did not show a direct correlation with a reduction in virus replication. However, mutant virus assembly was reduced in parallel with reduced virus replication. Additionally, a reassortant of strain A/WSN/33 (WSN), containing the Ud HA gene with mutations C555S, C562S, and C565S, produced virus that could form plaques on regular MDCK cells and had only moderately decreased replication, suggesting differences in the interactions between Ud and WSN HA and internal viral proteins. Analysis of M1 mutants containing substitutions in the six residues that differ between the Ud and WSN M1 proteins indicated that a constellation of residues are responsible for the difference between the M1 proteins in their ability to support virus assembly with nonpalmitoylated H3 HA.

scholarly journals Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
James D. Allen ◽  
Ted M. Ross
Keyword(s):  
Influenza Virus ◽  
Immune Responses ◽  
Influenza A ◽  
Seasonal Influenza ◽  
Influenza Viruses ◽  
Next Generation ◽  
Virus Infections ◽  
Wild Type ◽  
Influenza Hemagglutinin ◽  
H3n2 Influenza

AbstractWhile vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

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