On the Stability of Parainfluenza Virus 5 F Proteins

Poor, Taylor A.; Song, Albert S.; Welch, Brett D.; Kors, Christopher A.; Jardetzky, Theodore S.; Lamb, Robert A.

doi:10.1128/jvi.03221-14

Cited by 6 publications

(13 citation statements)

References 16 publications

(16 reference statements)

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“…Of particular interest was a highly destabilizing S443D mutation. As previously reported (38), cell-surface expression measured by prefusion-specific monoclonal F1a antibody binding (39) was not detected for F-S443D despite greater-than-W3A levels of fusion protein activity measured by a luciferase reporter assay and syncytia formation (Fig. 1).…”

Section: Resultssupporting

confidence: 51%

“…The stabilizing P22L mutation has been proposed to function through local interactions between the leucine side chain and a hydrophobic pocket on the side of the F-protein globular head, whereas the destabilizing S443D mutation has been suggested to function by disrupting a local hydrogen bond network at the base of the F-protein globular head (38) (Fig. 2A, Right and Left Inset, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…Along with the prefusion PIV5 WR F structure and characterization of the P22L mutation, we previously reported on destabilizing mutations at residue 443 (38). Of particular interest was a highly destabilizing S443D mutation.…”

Section: Resultsmentioning

confidence: 99%

“…All experiments were done in triplicate and error bars represent ±1 SD. S443P and S443D data were described previously (38). (Magnification: 160×.)…”

Section: Resultsmentioning

confidence: 99%

“…We subsequently performed site-directed mutagenesis to characterize the prefusion stabilization conferred by the P22L mutation (38). Although substitutions of residue 22 by hydrophobic amino acids smaller than leucine demonstrated W3A levels of fusion activity, hydrophobic side chains equal to or larger than leucine demonstrated diminished levels of fusion activity.…”

mentioning

confidence: 99%

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Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Song¹,

Poor²,

Abriata

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Parainfluenza virus 5 (PIV5) is an enveloped, single-stranded, negative-sense RNA virus of the Paramyxoviridae family. PIV5 fusion and entry are mediated by the coordinated action of the receptor-binding protein, hemagglutinin–neuraminidase (HN), and the fusion protein (F). Upon triggering by HN, F undergoes an irreversible ATP- and pH-independent conformational change, going down an energy gradient from a metastable prefusion state to a highly stable postfusion state. Previous studies have highlighted key conformational changes in the F-protein refolding pathway, but a detailed understanding of prefusion F-protein metastability remains elusive. Here, using two previously described F-protein mutations (S443D or P22L), we examine the capacity to modulate PIV5 F stability and the mechanisms by which these point mutants act. The S443D mutation destabilizes prefusion F proteins by disrupting a hydrogen bond network at the base of the F-protein globular head. The introduction of a P22L mutation robustly rescues destabilized F proteins through a local hydrophobic interaction between the N-terminal helix and a hydrophobic pocket. Prefusion stabilization conferred by a P22L-homologous mutation is demonstrated in the F protein of Newcastle disease virus, a paramyxovirus of a different genus, suggesting a conserved stabilizing structural element within the paramyxovirus family. Taken together, the available data suggest that movement of the N-terminal helix is a necessary early step for paramyxovirus F-protein refolding and presents a novel target for structure-based drug design.

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Section: Resultssupporting

confidence: 51%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…All experiments were done in triplicate and error bars represent ±1 SD. S443P and S443D data were described previously (38). (Magnification: 160×.)…”

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Song¹,

Poor²,

Abriata

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Renaissance of protein crystallization and precipitation in biopharmaceuticals purification

Santos

Carvalho

Roque

2017

Biotechnology Advances

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Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation

Abdella

Aggarwal

Okura

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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132

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Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ∼250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L–P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5′ end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

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On the Stability of Parainfluenza Virus 5 F Proteins

Cited by 6 publications

References 16 publications

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Immobilization of the N-terminal helix stabilizes prefusion paramyxovirus fusion proteins

Renaissance of protein crystallization and precipitation in biopharmaceuticals purification

Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation

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