A Conserved Region in the F
            <sub>2</sub>
            Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Gardner, Amanda E.; Dutch, Rebecca Ellis

doi:10.1128/jvi.00366-07

Cited by 34 publications

(38 citation statements)

References 59 publications

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“…In that study, pathogenesis was not tested, but fusoge- nicity of A2 F could be increased or decreased depending on the residue at position 66 (17). Based on this recent work along with our results and reports on other paramyxovirus proteins, the loop region of F 2 containing residue 79 appears to be involved in the mechanism of fusion (17,32,33). An individual residue in F 2 was implicated in the differential fusogenicity of wild-type measles virus compared to the Edmonston strain of measles virus (33).…”

Section: Discussionmentioning

confidence: 96%

“…An individual residue in F 2 was implicated in the differential fusogenicity of wild-type measles virus compared to the Edmonston strain of measles virus (33). In 2007, Gardner and Dutch demonstrated that in non-Pneumovirus paramyxoviruses, a conserved region in F 2 encompassing residue 79 drastically affected fusion activity in vitro (32). In addition, for human parainfluenza virus 5, they postulated a potential interaction of this region with heptad repeat A (32).…”

Section: Discussionmentioning

confidence: 99%

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Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Hotard

Lee

Currier

et al. 2015

J Virol

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Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein. IMPORTANCEHuman respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.H uman respiratory syncytial virus (hRSV according to the International Committee on Taxonomy of Viruses; often abbreviated RSV) is a negative-sense, nonsegmented, and singlestranded RNA virus in the Paramyxoviridae family. RSV is the most important viral lower respiratory tract pathogen for infants, causing 16 times more hospitalizations than influenza virus in children under 1 year old (1). There is currently no vaccine licensed to prevent RSV infections; there is also a need for additional therapies, and further knowledge of RSV pathogenesis will enhance efforts toward these goals.Strains of RSV exhibit differential pathogenesis in the BALB/c mouse model of RSV disease (2-4). A strain called line 19 was originally isolated in 1967 and then passaged in primary chick kidney cells, as well as primary chick lung cells, prior to 72 passages in human MRC-5 fibroblast cells (...

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Section: Discussionmentioning

confidence: 96%

Section: Discussionmentioning

confidence: 99%

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Hotard

Lee

Currier

et al. 2015

J Virol

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“…A specific histidine residue at the domain I to III interface has also been recently shown to be critical for the triggering of the type II tick-borne encephalitis E fusion protein (22). Specific regions in paramyxovirus F proteins have also been implicated in the modulation of fusion activity under neutral pH conditions (23,46), as the replacement of residues in these regions can increase or decrease fusion depending on the side chain present. One such region, referred to as the heptad repeat B (HRB) linker, is located N terminal to the C-terminal heptad repeat region (46).…”

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confidence: 99%

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

Schowalter

Chang

Robach

et al. 2009

J Virol

115

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Human metapneumovirus (HMPV) is a significant respiratory pathogen classified in the Pneumovirinae subfamily of the paramyxovirus family. Recently, we demonstrated that HMPV F protein-promoted cell-cell fusion is stimulated by exposure to low pH, in contrast to what is observed for other paramyxovirus F proteins. In the present study, we examined the potential role of histidine protonation in HMPV F fusion and investigated the role of low pH in HMPV viral entry. Mutagenesis of the three ectodomain histidine residues of the HMPV F protein demonstrated that the mutation of a histidine in the heptad repeat B linker domain (H435) ablated fusion activity without altering cell surface expression or proteolytic processing significantly. Modeling of the HMPV F protein revealed several basic residues surrounding this histidine residue, and the mutation of these residues also reduced fusion activity. These results suggest that electrostatic repulsion in the heptad repeat B linker region may contribute to the triggering of HMPV F. In addition, we examined the effect of inhibitors of endosomal acidification or endocytosis on the entry of a recombinant green fluorescent proteinexpressing HMPV. Interestingly, chemicals that raise the pH of endocytic vesicles resulted in a 30 to 50% decrease in HMPV infection, while the inhibitors of endocytosis reduced infection by as much as 90%. These data suggest that HMPV utilizes an endocytic entry mechanism, in contrast to what has been hypothesized for most paramyxoviruses. In addition, our results indicate that HMPV uses the low pH of the endocytic pathway to enhance infectivity, though the role of low pH likely differs from classically described mechanisms.

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“…Previously, an in vitro study indicated that a conserved region in the F2 subunit of paramyxoviruses (amino acid residues 49-88) plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion (Gardner & Dutch, 2007). In this region, there are two amino acid differences between the F2 subunits of strains BC and AKO.…”

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confidence: 99%

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation

Kim

Xiao

Collins

et al. 2016

Journal of General Virology

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The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.Virulent strains of Newcastle disease virus (NDV) cause a devastating disease in chickens leading to major economic losses in the poultry industry worldwide (Alexander, 1989). NDV is a member of the genus Avulavirus in the family Paramyxoviridae. The virion is enveloped, with a non-segmented, negative-sense ssRNA genome (Samal, 2011). The genome encodes a nucleocapsid protein (N), a phosphoprotein (P), a matrix protein (M), a fusion protein (F), a haemagglutinin-neuraminidase protein (HN) and a large polymerase protein (L).NDV isolates vary greatly in their pathogenicity for chickens and are categorized into three main pathotypes: lentogenic (avirulent), mesogenic (moderately virulent) and velogenic (highly virulent) (Alexander, 1989). The amino acid sequence at the F protein cleavage site has been identified as the primary determinant of virulence (Panda et al., 2004;Peeters et al., 1999). Virulent NDV strains have multibasic cleavage sequences that contain the preferred cleavage site of the intracellular protease furin (Arg-X-Arg/Lys-Arg # ), available in most cell types. In contrast, avirulent NDV strains typically contain one or two basic residues at the F protein cleavage site and are delivered to the plasma membrane in an uncleaved form for cleavage by extracellular proteases. Modification of the cleavage site sequence in the F protein has been shown to greatly alter the replication and pathogenicity of NDV (de Leeuw et al., 2005;Hu et al., 2011;Panda et al., 2004;Peeters et al., 1999; Xiao et al., 2012). In our previous study, we modified the F protein cleavage site sequence (RRQKR # F) of a highly virulent NDV strain Ban010 (genotype VII) to that of avirulent NDV strain LaSota (GRQGR # L), resulting in an avirulent derivative rBan/AF (Xiao et al., 2012). The parental virulent Ban010 virus produced extensive syncytia and plaque in cell culture in the absence of added protease, whereas the avirulent rBan/AF derivative caused only single-cell infections without syncytia or plaque formation in the presence or absence of extracellular protease. This result was unexpecte...

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A Conserved Region in the F ₂ Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Cited by 34 publications

References 59 publications

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation

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A Conserved Region in the F 2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Cited by 34 publications

References 59 publications

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis

Low-pH Triggering of Human Metapneumovirus Fusion: Essential Residues and Importance in Entry

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation

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A Conserved Region in the F ₂ Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation