Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

Webb, Stacy R.; Nagy, Tamas L.; Moseley, Hunter N. B.; Fried, Mike; Dutch, Rebecca Ellis

doi:10.1074/jbc.m117.777235

Cited by 14 publications

(26 citation statements)

References 54 publications

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“…Viral fusion proteins drive the fusion of viral and cellular membranes, a key early step in the entry of enveloped viruses. We have previously shown that the TM domains of several paramyxovirus fusion proteins associate in isolation and that disruption of TM-TM association by mutagenesis of a key L/I zipper motif resulted in a HeV F protein that triggered prematurely (11,18). Based on the previous studies, we examined the ability of paramyxovirus TM-TM interactions to be targeted with exogenous proteins containing the TM domain to cause disruption in overall protein structure and function (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…To facilitate fusion, the HeV F protein must be at the surface and in its cleaved (F 1 ϩF 2 ) form. Previous work has shown that disruption of the HeV TM interactions significantly lowered the amount of F protein expressed at the cell surface (18). To determine whether the F protein was trafficked to the cell surface in the presence of the exogenous TM proteins, the proteins were analyzed with a radiolabel surface biotinylation assay.…”

Section: Hev F Tm Proteins Interact With the Full-length Hev Fmentioning

confidence: 99%

“…There are several points in the early stages of the paramyxovirus fusion process that can be targeted for disruption: the receptor binding step mediated by the attachment protein, the interaction between the attachment protein and the fusion protein that facilitates triggering of F, and the overall refolding of the fusion protein necessary for membrane fusion (14). Class I fusion proteins, including those of the family Paramyxoviridae, are folded as trimers (15), and it has been shown that the TM domain is important for proper protein folding and function (5,6,9,12,(16)(17)(18). To drive membrane fusion, the fusion protein must undergo dramatic conformational rearrangements from the metastable prefusion conformation to the postfusion conformation.…”

mentioning

confidence: 99%

“…Previously, we have shown that the HeV F protein TM domain associates in a monomer-trimer equilibrium in isolation and contributes to overall F protein stability. More specifically, we have shown that HeV F TM-TM association is sequence specific, with a leucine/isoleucine (L/I) zipper motif significantly contributing to the interaction (10,18). The HeV F protein is synthesized as a trimer with a domain structure composed of two heptad repeat domains (HRA and HRB), a highly hydrophobic fusion peptide (FP), a TM domain, and a cytoplasmic tail (CT) ( Fig.…”

mentioning

confidence: 99%

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A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability

Barrett

Webb

Dutch

2019

J Virol

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Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the family Paramyxoviridae, utilizes the attachment protein (G) and the fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a large, irreversible conformation change to drive membrane fusion. Previously, we have shown that the transmembrane (TM) domain of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium. This TM-TM association contributes to the stability of the prefusion form of the protein, supporting a role for TM-TM interactions in the control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Coexpression of these constructs with HeV F resulted in dramatic reductions in the stability of F protein expression and fusion activity. In contrast, no effects were observed when the HeV F TM constructs were coexpressed with the nonhomologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited infection with PIV5 but did not significantly affect infection with human metapneumovirus, a related virus. These results indicate that targeted disruption of TM-TM interactions significantly impact viral fusion protein stability and function, presenting these interactions as a novel target for antiviral development. IMPORTANCE Enveloped viruses require virus-cell membrane fusion to release the viral genome and replicate. The viral fusion protein triggers from the pre-to the postfusion conformation, an essentially irreversible change, to drive membrane fusion. We found that small proteins containing the TM and a limited flanking region homologous to the fusion protein of the zoonotic Hendra virus reduced protein expression and fusion activity. The introduction of exogenous TM peptides may displace a TM domain, disrupting native TM-TM interactions and globally destabilizing the fusion protein. Supporting this hypothesis, we showed that a sequence-specific transmembrane peptide dramatically reduced viral infection in another enveloped virus model, suggesting a broader inhibitory mechanism. Viral fusion protein TM-TM interactions are important for protein function, and disruption of these interactions dramatically reduces protein stability.

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Section: Discussionmentioning

confidence: 99%

Section: Hev F Tm Proteins Interact With the Full-length Hev Fmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability

Barrett

Webb

Dutch

2019

J Virol

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“…The TM domain was subsequently shown to stabilize the F protein in its pre-fusion conformation [134]. A heptad repeat pattern of β-branched residues (e.g., leucine, isoleucine, threonine, valine) was identified to be highly conserved in the TM domains of 140 PMVs.…”

Section: Novel Functions Of the Pmv-f Transmembrane Domainmentioning

confidence: 99%

Differential Features of Fusion Activation within the Paramyxoviridae

Azarm

Lee

2020

Viruses

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Paramyxovirus (PMV) entry requires the coordinated action of two envelope glycoproteins, the receptor binding protein (RBP) and fusion protein (F). The sequence of events that occurs during the PMV entry process is tightly regulated. This regulation ensures entry will only initiate when the virion is in the vicinity of a target cell membrane. Here, we review recent structural and mechanistic studies to delineate the entry features that are shared and distinct amongst the Paramyxoviridae. In general, we observe overarching distinctions between the protein-using RBPs and the sialic acid- (SA-) using RBPs, including how their stalk domains differentially trigger F. Moreover, through sequence comparisons, we identify greater structural and functional conservation amongst the PMV fusion proteins, as compared to the RBPs. When examining the relative contributions to sequence conservation of the globular head versus stalk domains of the RBP, we observe that, for the protein-using PMVs, the stalk domains exhibit higher conservation and find the opposite trend is true for SA-using PMVs. A better understanding of conserved and distinct features that govern the entry of protein-using versus SA-using PMVs will inform the rational design of broader spectrum therapeutics that impede this process.

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Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

Barrett¹,

Neal²,

Edmonds³

et al. 2021

Journal of Biological Chemistry

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The trimeric SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and TMPRSS2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. Additionally, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high profile therapeutic target.

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Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability

Cited by 14 publications

References 54 publications

A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability

A Hydrophobic Target: Using the Paramyxovirus Fusion Protein Transmembrane Domain To Modulate Fusion Protein Stability

Differential Features of Fusion Activation within the Paramyxoviridae

Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

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