Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

Bose, Sayantan; Heath, Carissa M.; Shah, Priya A.; Alayyoubi, Maher; Jardetzky, Theodore S.; Lamb, Robert A.

doi:10.1128/jvi.02123-13

Cited by 54 publications

(63 citation statements)

References 61 publications

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“…Recent studies on PIV5 HN, NDV-HN, MeV H, and CDV H have implicated the F-interacting and F-activating regions to be in the central part of the HN or H stalk (29,33,41,43,44,56,60). Recently, we identified residues located within and near the PIV5-F "Iglike" domain as putative HN-interacting residues (61). Given that most of these identified residues were hydrophobic, the nature of corresponding F-interacting residues on the HN stalk and their roles in fusion activation were of great interest and have been investigated here.…”

Section: Resultsmentioning

confidence: 99%

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

Bose

Song

Jardetzky

et al. 2014

J Virol

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113

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Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion. HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirusmediated membrane fusion. IMPORTANCE Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [

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Section: Resultsmentioning

confidence: 99%

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

Bose

Song

Jardetzky

et al. 2014

J Virol

Self Cite

113

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“…3C). As domain II was previously identified as a critical site for F-activation by HN (49,50), F-S443D's increase in mechanical coupling between the N terminus and domain II may suggest a functionally critical interaction during the F-protein refolding pathway.…”

Section: Discussionmentioning

confidence: 99%

“…pCAGGS-F and pCAGGS-HN expression constructs harboring the PIV5 W3A F and HN or NDV F and HN (strain Australia-Victoria) genes were used as described previously (19,50). Mutations in pCAGGS-F were constructed by four-primer PCR.…”

Section: Methodsmentioning

confidence: 99%

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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“…A body of evidence suggests that distinct syncytia are formed by transiently expressed viral glycoproteins and viral entry (25)(26)(27)(28). To test the bioactivity of the F cysteines in the biologically relevant context of intact virions, we concentrated on the three most membrane-proximal constructs, L490C, M487C, and L484C, which were transported to the cell surface most efficiently, and individually replaced the F-protein-encoding ORF with the ORF of these mutants in a cDNA copy of the MeV genome.…”

Section: Recovery Of Mev Recombinants With Disulfide Bonds In F-protementioning

confidence: 99%

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

Brindley¹,

Plattet²,

Plemper³

2014

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

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Enveloped viruses such as HIV and members of the paramyxovirus family use metastable, proteinaceous fusion machineries to merge the viral envelope with cellular membranes for infection. A hallmark of the fusogenic glycoproteins of these pathogens is refolding into a thermodynamically highly stable fusion core structure composed of six antiparallel α-helices, and this structure is considered instrumental for pore opening and/or enlargement. Using a paramyxovirus fusion (F) protein, we tested this paradigm by engineering covalently restricted F proteins that are predicted to be unable to close the six-helix bundle core structure fully. Several candidate bonds formed efficiently, resulting in F trimers and higher-order complexes containing covalently linked dimers. The engineered F complexes were incorporated into recombinant virions efficiently and were capable of refolding into a postfusion conformation without temporary or permanent disruption of the disulfide bonds. They efficiently formed fusion pores based on virus replication and quantitative cell-to-cell and virus-to-cell fusion assays. Complementation of these F mutants with a monomeric, fusion-inactive F variant enriched the F oligomers for heterotrimers containing a single disulfide bond, without affecting fusion complementation profiles compared with standard F protein. Our demonstration that complete closure of the fusion core does not drive paramyxovirus entry may aid the design of strategies for inhibiting virus entry. membrane fusion | measles virus M embrane fusion is an essential process in eukaryotic cell biology. Examples range from fusion of lipid vesicles to sustain intracellular transport along secretory and endocytotic pathways to cell fusion in fertilization and development and to the fusion of viral with cellular membranes resulting in viral infection. Because there are several high-energy barriers (1), lipid membranes do not merge spontaneously under physiological conditions and therefore require the aid of auxiliary proteins to induce membrane stress and lower the activation energy for lipid merger. For vesicular transport, for instance, soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) proteins resident in both the target and donor membrane mediate the process (2). Two fundamental differences distinguish cellular fusion machineries from viral fusogenic membrane glycoproteins (vFMGs), which mediate the entry of enveloped viruses by the fusion of the envelope with cellular membranes: (i) vFMGs do not require adapter proteins but insert a fusion peptide domain into the target membrane; and (ii) vFMGs fold into metastable prefusion conformations, obviating the dependence on external energy sources. Three distinct types of viral fusion (F) proteins have been classified: class I vFMGs are found, among others, on influenza virus, retroviruses, and paramyxoviruses; class II proteins are present on alpha-and flaviviruses; and class III proteins mediate herpes-and rhabdovirus entry (reviewed in ref.3).Once the fusion p...

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Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

Cited by 54 publications

References 61 publications

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

Fusion Activation through Attachment Protein Stalk Domains Indicates a Conserved Core Mechanism of Paramyxovirus Entry into Cells

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

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

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