Cysteines Flanking the Internal Fusion Peptide Are Required for the Avian Sarcoma/Leukosis Virus Glycoprotein To Mediate the Lipid Mixing Stage of Fusion with High Efficiency

ABSTRACT We previously showed that the cysteines flanking the internal fusion peptide of the avian sarcoma/leukosis virus subtype A (ASLV-A) Env (EnvA) are important for infectivity and cell-cell fusion. Here we define the stage of fusion at which the cysteines are required. The flanking cysteines are dispensable for receptor-triggered membrane association but are required for the lipid mixing step of fusion, which, interestingly, displays a high pH onset and a biphasic profile. Second-site mutations that partially restore infection partially restore lipid mixing. These findings indicate that the cysteines flanking the internal fusion peptide of EnvA (and perhaps by analogy Ebola virus glycoprotein) are important for the foldback stage of the conformational changes that lead to membrane merger.

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A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

Journal of Virology ◽

10.1128/jvi.00526-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8575-8586 ◽

Cited By ~ 5

Author(s):

Deborah C. Melder ◽

Xueqian Yin ◽

Sue E. Delos ◽

Mark J. Federspiel

Keyword(s):

Conformational Changes ◽

Ebola Virus ◽

Specific Interaction ◽

Fusion Peptide ◽

Replication Capacity ◽

Cysteine Residues ◽

Complete Fusion ◽

Wild Type ◽

Site Mutation ◽

Avian Sarcoma

ABSTRACT The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

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The Avian Retrovirus Avian Sarcoma/Leukosis Virus Subtype A Reaches the Lipid Mixing Stage of Fusion at Neutral pH

Journal of Virology ◽

10.1128/jvi.77.5.3058-3066.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3058-3066 ◽

Cited By ~ 52

Author(s):

Laurie J. Earp ◽

Sue E. Delos ◽

Robert C. Netter ◽

Paul Bates ◽

Judith M. White

Keyword(s):

Cell Fusion ◽

Receptor Binding ◽

Low Ph ◽

Target Cells ◽

Dependent Manner ◽

Neutral Ph ◽

Subtype A ◽

Avian Sarcoma ◽

Virus Subtype ◽

Cell Cell

ABSTRACT We previously showed that the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.

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Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer

RSC Advances ◽

10.1039/c7ra02940c ◽

2017 ◽

Vol 7 (37) ◽

pp. 22741-22748 ◽

Cited By ~ 7

Author(s):

Nisha Bhattarai ◽

Jeevan B. GC ◽

Bernard S. Gerstman ◽

Robert V. Stahelin ◽

Prem P. Chapagain

Keyword(s):

Plasma Membrane ◽

Conformational Changes ◽

Ebola Virus ◽

Membrane Binding ◽

Marburg Virus ◽

Virus Protein ◽

Membrane Association ◽

Binding Interface

The membrane binding interface of the Marburg virus protein mVP40 dimer differs from that of the Ebola virus eVP40 dimer but membrane binding allows conformational changes in mVP40 that makes it structurally similar to the eVP40 dimer.

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Critical Role for the Cysteines Flanking the Internal Fusion Peptide of Avian Sarcoma/Leukosis Virus Envelope Glycoprotein

Journal of Virology ◽

10.1128/jvi.74.20.9738-9741.2000 ◽

2000 ◽

Vol 74 (20) ◽

pp. 9738-9741 ◽

Cited By ~ 32

Author(s):

S. E. Delos ◽

J. M. White

Keyword(s):

Cell Fusion ◽

Disulfide Bond ◽

Envelope Glycoprotein ◽

Ebola Virus ◽

Leukemia Virus ◽

Critical Role ◽

Fusion Peptide ◽

Virus Envelope ◽

Mediate Cell ◽

Avian Sarcoma

ABSTRACT The transmembrane subunit (TM) of the envelope glycoprotein (Env) of the oncovirus avian sarcoma/leukosis virus (ASLV) contains an internal fusion peptide flanked by two cysteines (C9 and C45). These cysteines, as well as an analogous pair in the Ebola virus GP glycoprotein, are predicted to be joined by a disulfide bond. To examine the importance of these cysteines, we mutated C9 and C45 in the ASLV subtype A Env (EnvA), individually and together, to serine. All of the mutant EnvAs formed trimers that were composed of the proteolytically processed surface (SU) and TM subunits. All mutant EnvAs were incorporated into murine leukemia virus pseudotyped virions and bound receptor with wild-type affinity. Nonetheless, all mutant EnvAs were significantly impaired (∼1,000-fold) in their ability to support infectivity. They were also significantly impaired in their ability to mediate cell-cell fusion. Our data are consistent with a model in which the internal fusion peptide of ASLV-A EnvA exists as a loop that is stabilized by a disulfide bond at its base and in which this stabilized loop serves an important function during virus-cell fusion. The fusion peptide of the Ebola virus GP glycoprotein may conform to a similar structure.

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Faculty Opinions recommendation of Endosomal proteolysis of the Ebola virus glycoprotein is necessary for infection.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1026055.322926 ◽

2005 ◽

Author(s):

Gabriella Campadelli-Fiume

Keyword(s):

Ebola Virus ◽

Virus Glycoprotein

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The Roles of Histidines and Charged Residues as Potential Triggers of a Conformational Change in the Fusion Loop of Ebola Virus Glycoprotein

PLoS ONE ◽

10.1371/journal.pone.0152527 ◽

2016 ◽

Vol 11 (3) ◽

pp. e0152527 ◽

Cited By ~ 4

Author(s):

Jinwoo Lee ◽

Sonia M. Gregory ◽

Elizabeth A. Nelson ◽

Judith M. White ◽

Lukas K. Tamm

Keyword(s):

Conformational Change ◽

Ebola Virus ◽

Virus Glycoprotein ◽

Charged Residues ◽

Fusion Loop

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Ebola virus glycoprotein directly triggers T lymphocyte death despite of the lack of infection

PLoS Pathogens ◽

10.1371/journal.ppat.1006397 ◽

2017 ◽

Vol 13 (5) ◽

pp. e1006397 ◽

Cited By ~ 27

Author(s):

Mathieu Iampietro ◽

Patrick Younan ◽

Andrew Nishida ◽

Mukta Dutta ◽

Ndongala Michel Lubaki ◽

...

Keyword(s):

T Lymphocyte ◽

Ebola Virus ◽

Virus Glycoprotein

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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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