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

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.

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

Journal of Virology ◽

10.1128/jvi.74.16.7529-7537.2000 ◽

2000 ◽

Vol 74 (16) ◽

pp. 7529-7537 ◽

Cited By ~ 42

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.

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

Journal of Virology ◽

10.1128/jvi.79.10.6449-6458.2005 ◽

2005 ◽

Vol 79 (10) ◽

pp. 6449-6458 ◽

Cited By ~ 65

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.

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The Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein: Analysis of Transmembrane Domain Length and Sequence Requirements

Journal of Virology ◽

10.1128/jvi.02252-08 ◽

2009 ◽

Vol 83 (9) ◽

pp. 4447-4461 ◽

Cited By ~ 17

Author(s):

Zhaofei Li ◽

Gary W. Blissard

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Membrane Fusion ◽

Autographa Californica ◽

Single Amino Acid ◽

Cell Surface Expression ◽

Virus Budding ◽

C Terminus ◽

Tm Domain ◽

Sequence Requirements

ABSTRACT GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. Previous transmembrane (TM) domain replacement studies showed that the TM domain serves a critical role in GP64 function. To extend the prior studies and examine specific sequence requirements of the TM domain, we generated a variety of GP64 TM domain mutations. The mutations included 4- to 8-amino-acid deletions, as well as single and multiple point mutations. While most TM domain deletion constructs remained fusion competent, those containing deletions of eight amino acids from the C terminus did not mediate detectable fusion. The addition of a hydrophobic amino acid (A, L, or V) to the C terminus of construct C8 (a construct that contains a TM domain deletion of eight amino acids from the C terminus) restored fusion activity. These data suggest that the membrane fusion function of GP64 is dependent on a critical length of the hydrophobic TM domain. All GP64 proteins with a truncated TM domain mediated detectable virion budding with dramatically lower levels of efficiency than wild-type GP64. The effects of deletions of various lengths and positions in the TM domain were also examined for their effects on viral infectivity. Further analysis of the TM domain by single amino acid substitutions and 3-alanine scanning mutations identified important but not essential amino acid positions. These studies showed that amino acids at positions 485 to 487 and 503 to 505 are important for cell surface expression of GP64, while amino acids at positions 483 to 484 and 494 to 496 are important for virus budding. Overall, our results show that specific features and amino acid sequences, particularly the length of the hydrophobic TM domain, play critical roles in membrane anchoring, membrane fusion, virus budding, and infectivity.

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A single amino acid deletion at the amino terminus of influenza virus hemagglutinin causes malfolding and blocks exocytosis of the molecule in mammalian cells.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)45855-3 ◽

1992 ◽

Vol 267 (4) ◽

pp. 2142-2148

Author(s):

C C Chao

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Mammalian Cells ◽

Single Amino Acid ◽

Amino Terminus ◽

Amino Acid Deletion ◽

Influenza Virus Hemagglutinin

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Human endothelin receptor ETB. Amino acid sequence requirements for super stable complex formation with its ligand.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)37315-5 ◽

1994 ◽

Vol 269 (10) ◽

pp. 7509-7513

Author(s):

T. Takasuka ◽

T. Sakurai ◽

K. Goto ◽

Y. Furuichi ◽

T. Watanabe

Keyword(s):

Amino Acid ◽

Complex Formation ◽

Amino Acid Sequence ◽

Stable Complex ◽

Endothelin Receptor ◽

Sequence Requirements

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New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

Journal of Virology ◽

10.1128/jvi.76.9.4456-4466.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4456-4466 ◽

Cited By ~ 42

Author(s):

Jennifer A. Gruenke ◽

R. Todd Armstrong ◽

William W. Newcomb ◽

Jay C. Brown ◽

Judith M. White

Keyword(s):

Influenza Virus ◽

Conformational Change ◽

Conformational Changes ◽

Limited Proteolysis ◽

Coiled Coil ◽

Fusion Peptide ◽

Wild Type ◽

Influenza Virus Hemagglutinin ◽

Target Membrane ◽

Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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