Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Mike; Dutch, Rebecca Ellis

doi:10.1074/jbc.m113.514554

Cited by 35 publications

(58 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Also, packaging of a heterologous glycoprotein may result in attenuation of the vector. Additional effects may occur: for example, evidence from several paramyxoviruses implicated the TM, CT, and other domains in modulating fusogenic activity, stability, and protein folding (17,19).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

Liang

Ngwuta

Herbert

et al. 2016

J Virol

View full text Add to dashboard Cite

Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499 -9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10-to 100-fold) and rhesus monkeys (100-to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8-and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. IMPORTANCEHuman respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this welltolerated vaccine candidate and should be applicable to other vectored vaccines.

show abstract

Section: Discussionmentioning

confidence: 99%

“…During viral assembly, the CT is thought to interact with internal virion proteins to mediate packaging of the F protein into virions (16)(17)(18). Evidence from several paramyxoviruses implicated TM and CT in modulating fusogenic activity, stability, and protein folding (17,19).…”

mentioning

confidence: 99%

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

Liang

Ngwuta

Herbert

et al. 2016

J Virol

View full text Add to dashboard Cite

show abstract

“…For example, self-assembly of paramyxovirus (43,44), influenza virus (45,46), and HIV (47) F-protein transmembrane domains was demonstrated experimentally and is considered a determinant for proper protein folding and fusion activity (44,47). Studies with class I vFMGs have shown that insertion of synthetic, soluble fusion peptides into the membrane can deform the lipid bilayer (48) and induce membrane fusion (49).…”

Section: Discussionmentioning

confidence: 99%

“…Vero-SLAM effector cells were transfected with plasmids encoding the DSP 1-7 dual-split protein (44), MeV H, and a mixture of F-encoding plasmids at the indicated ratios. Vero-SLAM target cells received plasmid DNA encoding the DSP 8-11 subunit.…”

Section: Methodsmentioning

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.

View full text Add to dashboard Cite

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...

show abstract

“…The F protein then undergoes a coordinated series of conformational changes to its most stable form to promote membrane fusion (17)(18)(19)(20). Thus, binding of the attachment glycoprotein to the cellular receptor(s) is critical for triggering the fusion process (21,22).…”

mentioning

confidence: 99%

Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection

Yun

Guan

Liu

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

Avian metapneumovirus (aMPV) fusion (F) protein mediates virus-cell membrane fusion to initiate viral infection, which requires F protein binding to its receptor(s) on the host cell surface. However, the receptor(s) for aMPV F protein is still not identified. All known subtype B aMPV (aMPV/B) F proteins contain a conserved Arg-Asp-Asp (RDD) motif, suggesting that the aMPV/B F protein may mediate membrane fusion via the binding of RDD to integrin. When blocked with integrin-specific peptides, aMPV/B F protein fusogenicity and viral replication were significantly reduced. Specifically we identified integrin ␣v and/or ␤1-mediated F protein fusogenicity and viral replication using antibody blocking, small interfering RNAs (siRNAs) knockdown, and overexpression. Additionally, overexpression of integrin ␣v and ␤1 in aMPV/B non-permissive cells conferred aMPV/B F protein binding and aMPV/B infection. When RDD was altered to RAE (Arg-Ala-Glu), aMPV/B F protein binding and fusogenic activity were profoundly impaired. These results suggest that integrin ␣v␤1 is a functional receptor for aMPV/B F protein-mediated membrane fusion and virus infection, which will provide new insights on the fusogenic mechanism and pathogenesis of aMPV. Avian metapneumovirus (aMPV)3 belongs to the genus Metapneumovirus in the subfamily Pneumovirinae of paramyxoviridae (1, 2). aMPV is a major cause of acute rhinotracheitis in turkeys and is associated with swollen head syndrome in chickens. Thus, aMPV is considered a major threat to the poultry industry (1, 2). Based on phylogenetic analysis, aMPV has been divided into four subtypes: aMPV/A, aMPV/B, aMPV/C, and aMPV/D (3).The entry of a paramyxovirus into the host cell initially requires fusion of the viral envelope and cellular membrane, which is mediated by the glycoproteins on the viral envelope (4 -6). For viruses of the Paramyxovirinae subfamily, such as Newcastle disease virus, fusion (F) protein-mediated membrane fusion requires the assistance of the hemagglutininneuraminidase (HN) protein (7-10). However, viruses in the Pneumovirinae subfamily including human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), and aMPV, the F protein alone in the absence of the small hydrophobic and attachment (G) proteins can mediate membrane fusion and viral infection (11-15), suggesting that the mechanism by which hRSV, hMPV, and aMPV mediate virus-cell membrane fusion might be unique among the Paramyxovirinae subfamily.The F protein of Paramyxovirinae viruses is activated by a non-F attachment glycoprotein bound to its receptor(s) on the host cell (10, 16). The F protein then undergoes a coordinated series of conformational changes to its most stable form to promote membrane fusion (17-20). Thus, binding of the attachment glycoprotein to the cellular receptor(s) is critical for triggering the fusion process (21,22). For example, Newcastle disease virus HN protein attaches to sialic acid-containing receptors on the host cell surface and then activates the F protein to induce membrane fu...

show abstract

Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

Cited by 35 publications

References 68 publications

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

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

Integrin αvβ1 Modulation Affects Subtype B Avian Metapneumovirus Fusion Protein-mediated Cell-Cell Fusion and Virus Infection

Contact Info

Product

Resources

About