Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion

The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses.     

Individual N-Glycans Added at Intervals along the Stalk of the Nipah Virus G Protein Prevent Fusion but Do Not Block the Interaction with the Homologous F Protein

The promotion of membrane fusion by most paramyxoviruses requires an interaction between the viral attachment and fusion (F) proteins to enable receptor binding by the former to trigger the activation of the latter for fusion. Numerous studies demonstrate that the F-interactive sites on the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) and measles virus (MV) hemagglutinin (H) proteins reside entirely within the stalk regions of those proteins. Indeed, stalk residues of NDV HN and MV H that likely mediate the F interaction have been identified. However, despite extensive efforts, the F-interactive site(s) on the Nipah virus (NiV) G attachment glycoprotein has not been identified. In this study, we have introduced individual N-linked glycosylation sites at several positions spaced at intervals along the stalk of the NiV G protein. Five of the seven introduced sites are utilized as established by a retardation of electrophoretic mobility. Despite surface expression, ephrinB2 binding, and oligomerization comparable to those of the wild-type protein, four of the five added N-glycans completely eliminate the ability of the G protein to complement the homologous F protein in the promotion of fusion. The most membrane-proximal added N-glycan reduces fusion by 80%. However, unlike similar NDV HN and MV H mutants, the NiV G glycosylation stalk mutants retain the ability to bind F, indicating that the fusion deficiency of these mutants is not due to prevention of the G-F interaction. These findings suggest that the G-F interaction is not mediated entirely by the stalk domain of G and may be more complex than that of HN/H-F.     

A dual-functional paramyxovirus F protein regulatory switch segment: activation and membrane fusion

Many viral fusion-mediating glycoproteins couple alpha-helical bundle formation to membrane merger, but have different methods for fusion activation. To study paramyxovirus-mediated fusion, we mutated the SV5 fusion (F) protein at conserved residues L447 and I449, which are adjacent to heptad repeat (HR) B and bind to a prominent cavity in the HRA trimeric coiled coil in the fusogenic six-helix bundle (6HB) structure. These analyses on residues L447 and I449, both in intact F protein and in 6HB, suggest a metamorphic region around these residues with dual structural roles. Mutation of L447 and I449 to aliphatic residues destabilizes the 6HB structure and attenuates fusion activity. Mutation of L447 and I449 to aromatic residues also destabilizes the 6HB structure despite promoting hyperactive fusion, indicating that 6HB stability alone does not dictate fusogenicity. Thus, residues L447 and I449 adjacent to HRB in paramyxovirus F have distinct roles in fusion activation and 6HB formation, suggesting this region is involved in a conformational switch.     

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

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.     

Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering.     

Importance of the cytoplasmic tails of the measles virus glycoproteins for fusogenic activity and the generation of recombinant measles viruses

The generation of replication-competent measles virus (MV) depends on the incorporation of biologically active, fusogenic glycoprotein complexes, which are required for attachment and penetration into susceptible host cells and for direct virus spread by cell-to-cell fusion. Whereas multiple studies have analyzed the importance of the ectodomains of the MV glycoproteins hemagglutinin (H) and fusion protein (F), we have investigated the role of the cytoplasmic tails of the F and H proteins for the formation of fusogenic complexes. Deletions in the cytoplasmic tails of transiently expressed MV glycoproteins were found to have varying effects on receptor binding, fusion, or fusion promotion activity. F tail truncation to only three amino acids did not affect fusion capacity. In contrast, truncation of the H cytoplasmic tail was limited. H protein mutants with cytoplasmic tails of <14 residues no longer supported F-mediated cell fusion, predominantly due to a decrease in surface expression and receptor binding. This indicates that a minimal length of the H protein tail of 14 amino acids is required to ensure a threshold local density to have sufficient accumulation of fusogenic H-F complexes. By using reverse genetics, a recombinant MV with an F tail of three amino acids (rMV-FcDelta30), as well as an MV with an H tail of 14 residues (rMV-HcDelta20), could be rescued, whereas generation of viruses with shorter H tails failed. Thus, glycoprotein truncation does not interfere with the successful generation of recombinant MV if fusion competence is maintained.     

Functional interaction between paramyxovirus fusion and attachment proteins

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share approximately 95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable.     

Localization of a Region in the Fusion Protein of Avian Metapneumovirus That Modulates Cell-Cell Fusion

The genus Metapneumovirus within the subfamily Pneumovirinae of the family Paramyxoviridae includes two members, human metapneumovirus (hMPV) and avian metapneumovirus (aMPV), causing respiratory tract infections in humans and birds, respectively. Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Membrane fusion of hMPV appears to be unique, in that fusion of some hMPV strains requires low pH. Here, we show that the fusion (F) proteins of aMPV promote fusion in the absence of the attachment protein and low pH is not required. Furthermore, there are notable differences in cell-cell fusion among aMPV subtypes. Trypsin was required for cell-cell fusion induced by subtype B but not subtypes A and C. The F protein of aMPV subtype A was highly fusogenic, whereas those from subtypes B and C were not. By construction and evaluation of chimeric F proteins composed of domains from the F proteins of subtypes A and B, we localized a region composed of amino acid residues 170 to 338 in the F protein that is responsible for the hyperfusogenic phenotype of the F from subtype A. Further mutagenesis analysis revealed that residues R295, G297, and K323 in this region collectively contributed to the hyperfusogenicity. Taken together, we have identified a region in the aMPV F protein that modulates the extent of membrane fusion. A model for fusion consistent with these data is presented.     

Residues of the human metapneumovirus fusion (F) protein critical for its strain-related fusion phenotype: implications for the virus replication cycle

The paramyxovirus F protein promotes fusion of the viral and cell membranes for virus entry, as well as cell-cell fusion for syncytium formation. Most paramyxovirus F proteins are triggered at neutral pH to initiate membrane fusion. Previous studies, however, demonstrated that human metapneumovirus (hMPV) F proteins are triggered at neutral or acidic pH in transfected cells, depending on the strain origin of the F sequences (S. Herfst et al., J. Virol. 82:8891-8895, 2008). We now report an extensive mutational analysis which identifies four variable residues (294, 296, 396, and 404) as the main determinants of the different syncytial phenotypes found among hMPV F proteins. These residues lie near two conserved histidines (H368 and H435) in a three-dimensional (3D) model of the pretriggered hMPV F trimer. Mutagenesis of H368 and H435 indicates that protonation of these histidines (particularly His435) is a key event to destabilize the hMPV F proteins that require low pH for cell-cell fusion. The syncytial phenotypes were reproduced in cells infected with the corresponding hMPV strains. However, the low-pH dependency for syncytium formation could not be related with a virus entry pathway dependent on an acidic environment. It is postulated that low pH may be acting for some hMPV strains as certain destabilizing mutations found in unusual strains of other paramyxoviruses. In any case, the results presented here and those reported by Schowalter et al. (J. Virol. 83:1511-1522, 2009) highlight the relevance of certain residues in the linker region and domain II of the pretriggered hMPV F protein for the process of membrane fusion.     

A Mutation in the Stalk of the Newcastle Disease Virus Hemagglutinin-Neuraminidase (HN) Protein Prevents Triggering of the F Protein despite Allowing Efficient HN-F Complex Formation

Newcastle disease virus (NDV)-induced membrane fusion requires formation of a complex between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Substitutions for NDV HN stalk residues A89, L90, and L94 block fusion by modulating formation of the HN-F complex. Here, we demonstrate that a nearby L97A substitution, though previously shown to block fusion, allows efficient HN-F complex formation and likely acts by preventing changes in the HN stalk required for triggering of the bound F protein.     

Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.     

Side Chain Packing below the Fusion Peptide Strongly Modulates Triggering of the Hendra Virus F Protein

Triggering of the Hendra virus fusion (F) protein is required to initiate the conformational changes which drive membrane fusion, but the factors which control triggering remain poorly understood. Mutation of a histidine predicted to lie near the fusion peptide to alanine greatly reduced fusion despite wild-type cell surface expression levels, while asparagine substitution resulted in a moderate restoration in fusion levels. Slowed kinetics of six-helix bundle formation, as judged by sensitivity to heptad repeat B-derived peptides, was observed for all H372 mutants. These data suggest that side chain packing beneath the fusion peptide is an important regulator of Hendra virus F triggering.Hendra virus and Nipah virus are highly pathogenic paramyxoviruses infecting humans. They were identified in 1994 and 1999, respectively, as the etiological agents behind cases of severe encephalitis and respiratory disease in Australia and Malaysia (7, 10, 17-18). Owing to their unusually high virulence, broad host range, and genetic similarity, Hendra virus and Nipah virus (NiV) have been classified into the new genus Henipavirus (31). Henipavirus membrane fusion requires the concerted effort of two viral surface glycoproteins (3-4, 30): the attachment protein (G), which binds receptor, and the fusion (F) protein, which drives membrane merger through vast conformational changes. Paramyxovirus F proteins are synthesized as inactive F₀ precursors which are subsequently cleaved into fusogenic disulfide-linked heterodimers, F₁+F₂. Despite a conserved requirement for cleavage, protease usage varies among paramyxoviruses, with henipavirus F being cleaved by the endosomal/lysosomal cysteine protease cathepsin L (19-20). This cleavage event positions the fusion peptide (FP) at the newly created N terminus and acts to prime the F protein. Following cleavage, the primed F protein must be triggered to begin the sequence of conformational changes required for membrane fusion. Like most F proteins, triggering of the henipavirus F proteins likely involves the henipavirus attachment proteins, though the mechanism remains poorly understood (reviewed in reference 28). F triggering facilitates refolding and extension of heptad repeat A (HRA) toward the target cell membrane, resulting in FP insertion into the bilayer (2). Further rearrangement brings HRA and HRB into close proximity, resulting in the formation of a stable six-helix bundle and culminating in a fully formed fusion pore (reviewed in reference 32).Cathepsin L cleavage of F does not require specific residues upstream of or at the cleavage site (K109) itself (5, 16), and the mechanism by which cathepsin L recognizes and specifically cleaves F is unclear. Modeling of the Hendra virus F amino acid sequence onto the prefusion structure of parainfluenza virus 5 (PIV5) (34) indicates that two of the three ectodomain histidine residues (H102 and H372) are positioned near the cathepsin L cleavage site following residue K109 (Fig. ​(Fig.11 A). In the monomer, H372 is located distally from K109, yet trimerization places H372 from one monomer directly beneath the FP and cleavage site of the neighboring monomer (Fig. ​(Fig.1A,1A, inset). We hypothesized that protonation of histidine residues could cause local conformational changes, potentially modulating cathepsin L cleavage, though these hypothesized conformational changes would not be due to direct modulation of K109 interactions since the predicted distances from K109 to either H102 or H372 are 12 Å and 27 Å, respectively (α-carbon to α-carbon distances). To test the role of H102 and H372 in cathepsin L cleavage, each was mutated individually or together to alanine (A) or asparagine (N), which has a side chain volume similar to that of histidine. Surface expression levels and cleavage of wild-type (WT) and mutant F proteins were examined by cell surface biotinylation as previously described (8). All mutant F proteins were surface expressed and cleaved, though levels of the H102A/H372A (AA) and H102N/H372N (NN) proteins were decreased compared to those of the wild type (Fig. ​(Fig.1B).1B). To examine cleavage kinetics, Vero cells transiently transfected with wild-type or mutant pCAGGS-Hendra virus F were metabolically labeled for 30 min and chased for 0 to 24 h. Band density corresponding to F₀ and F₁ was quantitated, and percent cleavage was defined as the density of F₁/(F₁+F₀). Cleavage kinetics of all Hendra virus F mutants were not significantly different from wild-type levels (Fig. ​(Fig.2).2). These data suggest that protonation of histidines in the region of the cleavage site is not involved in cathepsin L processing of Hendra virus F.Open in a separate windowFIG. 1.Structural modeling and surface expression of Hendra virus F H102 and H372 mutants. (A) Homology model of the Hendra virus F monomer based on the crystal structure of PIV5 F, shown as a ribbon diagram (image generated using PyMOL; Delano Scientific [www.pymol.org]): red, H102 and H372; blue, fusion peptide (FP); green, P1 cleavage site residue K109. The locations of H102 and H372 in the trimeric protein are shown in the box insert using the same color scheme except with an additional monomer shown in teal. (B) Surface expression of transiently transfected wild-type and Hendra virus F mutants in Vero cells following metabolic labeling (3 h). Surface proteins were biotinylated prior to immunoprecipitation, and the total and surface populations were separated by streptavidin pull-down. Proteins were analyzed via 15% SDS-PAGE and visualized using autoradiography. F₁ band quantitation via densitometry is shown normalized to wild-type levels plus or minus one standard deviation. The surface expression levels represent the averages of data from three independent experiments, with one representative gel shown.Open in a separate windowFIG. 2.Total protein cleavage time points for WT and mutant F protein. Vero cells transiently transfected with wild-type or mutant Hendra virus F were metabolically labeled (3 h) and chased for the indicated times. The total protein population was immunoprecipitated and analyzed by 15% SDS-PAGE and autoradiography. Band intensity was quantitated using the ImageQuant 5.2 software program (GE Healthcare, Piscataway, NJ), and percent cleavage was defined as the intensity of F₁/(F₁+F₀). Error bars are plus or minus one standard deviation and represent the average of data from three independent experiments.Since the mutants were expressed on the cell surface in the mature, cleaved form, fusion was examined using a syncytium assay (Fig. ​(Fig.33 A). Mutations at H102 did not significantly alter syncytium formation, but large reductions in fusion were observed for F proteins containing an H372 mutation. To quantitatively analyze fusion, a reporter gene assay was utilized. Since the single mutations all resulted in increased surface density (Fig. ​(Fig.1B)1B) while decreases were observed for the double mutants, analysis of the effects of surface density on WT Hendra virus F fusion was first performed. Previous work with other class I viral fusion proteins, including PIV5 F and influenza virus hemagglutinin (HA), has shown that surface density correlates with the final extent of fusion over a range of densities (6). Aguilar et al. (1) demonstrated that increasing the amount of NiV G and NiV F DNA transfected results in an increase in syncytium formation. However, a direct correlation between F surface expression and fusion has not been previously reported for henipaviruses. To assess this, Vero cells were transfected with various amounts of wild-type pCAGGS-Hendra virus F and biotinylated as described previously (8); reporter gene analyses using the same DNA amounts were performed alongside the biotinylation experiments. Increased surface densities clearly led to increases in fusion, though the correlation was not completely linear (Fig. ​(Fig.3B).3B). These data were then utilized to generate a percent WT fusion level for each mutant normalized for the observed cell surface expression levels. Mutations at H102 did not significantly alter fusion (Fig. ​(Fig.3C).3C). However, cell-cell fusion was dramatically reduced (10% to 20% of wild-type values) with the H372A and AA mutant F proteins. A partial restoration in fusion was seen for the H372N and NN mutants, suggesting that side chain volume plays a role; however, fusion levels were only 30% to 60% of those of the wild type (Fig. ​(Fig.3C).3C). While histidine residues proximal to the influenza virus HA fusion peptide have been shown to regulate low-pH triggering, Hendra virus F-mediated fusion occurs at neutral pH, and incubation at low pH has no effect on fusogenicity (A. Chang and R. E. Dutch, unpublished results). These data indicate that mutations at H372 result in a hypofusogenic protein, suggesting that side chain packing within this region may strongly modulate F protein triggering, potentially by altering protein stability.Open in a separate windowFIG. 3.Syncytium assay, reporter gene analysis, and correlation of wild-type and mutant F protein surface expression versus fusion activity. (A) Representative syncytium images from three independent experiments for wild-type and mutant Hendra virus F proteins. Vero cells transiently transfected with wild-type pCAGGS-Hendra virus G and wild-type or mutant pCAGGS-Hendra virus F were kept at 37°C for 24 to 48 h posttransfection, and photographs were taken using a Nikon digital camera mounted atop a Nikon TS100 microscope with a 10× objective. (B) Correlation between surface expression and fusogenicity for wild-type Hendra virus F. Vero cells transiently transfected with various amounts of wild-type Hendra virus F DNA were biotinylated, and reporter gene analysis was performed simultaneously using the same DNA quantities. (C) Reporter gene analysis of equal μg of wild-type or mutant Hendra virus F in pCAGGS normalized to average cell surface expression levels. Vero cells transiently transfected with wild-type Hendra virus G, wild-type or mutant Hendra virus F, and T7 luciferase were overlaid with BSR cells 18 h posttransfection, lysed, and assayed for luciferase activity (n = 5 to 8; ±95% confidence interval [CI]).The hypofusogenic phenotype of the H372 mutants could be explained by changes in the stability of the prefusion form following cleavage, resulting in altered fusion kinetics. To examine fusion kinetics, sensitivity over time to peptides which block formation of the postfusion six-helix bundle was examined (NiV C2; corresponding to HRB of the highly homologous Nipah virus F protein; generously provided by Chris Broder [Uniformed Services University of the Health Sciences]). One hundred nM NiV C2 has been shown to inhibit Henipavirus F-mediated cell-cell fusion (4). Similar peptides inhibit many other class I fusion proteins (11-13, 23, 33, 35-36). Vero cells transfected with wild-type pCAGGS-Hendra virus F and G and T7 luciferase were overlaid with target BSR cells on ice for 1 h. Prewarmed Dulbecco''s modified Eagle medium (DMEM) was added to initiate fusion, and at the indicated time points, DMEM containing 100 nM NiV C2 or 100 nM NiV SC (scrambled control peptide) was added. Cells were kept at 37°C for 3 h, and luciferase activity was assayed (Fig. ​(Fig.44 A). Cell-cell fusion kinetics for the wild-type Hendra virus F protein showed a steep increase in membrane fusion events from the 5- to 20-min time points (Fig. ​(Fig.4B,4B, solid line). Approximately 50% of cell-cell fusion events became insensitive to the addition of NiV C2 by 30 min, with the majority of membrane fusion events complete by 60 min (Fig. ​(Fig.4B,4B, solid line). Fusion kinetics for the H102A and H102N proteins closely resembled wild-type kinetics, consistent with overall fusion levels (Fig. 4B and C). In contrast, cell-cell fusion was dramatically slowed for F proteins containing mutations at H372. For all H372 mutants, no fusion was observed during the first 30 min, in stark contrast to results for the wild type. After 30 min, fusion was observed for the H372N and NN proteins, which reach 20 to 40% of maximal fusion within 60 min (Fig. ​(Fig.4C).4C). The small amount of fusion observed for the H372A protein occurred long after fusion was complete for the wild-type protein. Combined, these data demonstrate that mutation of H372 to either alanine or asparagine decreases the initial rate of membrane fusion potentially by increasing the energetic barrier for Hendra virus F triggering.Open in a separate windowFIG. 4.Cell-cell reporter gene fusion kinetics for wild-type and mutant Hendra virus F proteins. (A) Diagram of the experimental setup: a, binding of BSR cells; b, addition of DMEM, NiV-SC peptide, or NiV-C2 peptides (0-min time point); c, addition of NiV-C2 at indicated time points; d, continued incubation of cells. (B and C) Cell-cell fusion kinetics for wild-type and mutant F proteins. Reporter gene analysis was performed as described above following binding of BSR cells at 4°C, addition of either DMEM, NiV-SC, or NiV-C2 at the indicated times, and continued incubation for 3 h at 37°C. Percent maximal fusion is defined as the amount of fusion which occurred during a given time point as a fraction of membrane fusion for a given construct over the duration of the experiment (3 h) in the absence of any peptide. Error bars are 95% confidence intervals (n = 3 to 6).While most paramyxovirus F proteins, including the Hendra virus F protein, are thought to be triggered by specific interactions with a homotypic attachment protein (reviewed in reference 28), mutations within paramyxovirus F proteins which alter stability of the prefusion form (8, 15, 21-22, 27) can also strongly modulate triggering. H372 is modeled to be near a conserved domain, termed CBF₁, in the Hendra virus F prefusion structure. Studies suggest that CBF₁, which is structurally composed of β-sheets, is important for F protein folding (9), likely playing a critical role in stabilization of the fusion peptide following proteolytic cleavage, with the CBF₁ domain from one monomer interacting with the fusion peptide from the neighboring monomer. Mutations in CBF₁ in Hendra virus F resulted in folding defects which could not be rescued at decreased temperatures. Given the proximity of H372 to CBF₁ in Hendra virus F, changes in side chain packing could stabilize the fusion peptide following cleavage and thus decrease the ability of the protein to trigger efficiently. In the model structure, H372 is predicted to be surrounded by polar and nonpolar residues (within 5 Å of the side chain), including I425, N423, Q342, F376, and two FP residues, A125 and T129. Extension out to 10 Å reveals that H372 is also near four additional FP residues (A126, I128, T129, and V132), suggesting that substitution of H372 with a smaller residue (H372A) could alter the packing depth of the more C-terminal portion of the FP following cleavage.Studies from other systems also implicate the fusion peptide and surrounding residues as regulators of F-promoted membrane fusion (14, 24, 26, 29). The paramyxovirus fusion peptide is an important regulator of triggering, since conserved glycine residues within the FP have been shown to play a role in regulation and activation of F (26). Numerous mutations within the fusion peptide pocket of H5N1 influenza virus HA were shown to regulate the pH needed for HA activation (25), with only one mutation causing significant changes to HA expression or cleavage. Similar experiments using the H3 subtype of influenza virus HA also demonstrated changes in pH requirements upon mutation of certain fusion peptide-proximal residues (29). While influenza virus HA requires low pH for fusion promotion, the data presented here show that regulation of interactions with and around the fusion peptide is also critically important for triggering and fusion promotion of neutral-pH fusing systems. Thus, the decrease in the rate of triggering observed for the H372A mutant is consistent with a model in which residues surrounding the fusion peptide can act to regulate F-mediated fusion promotion independently of large changes in protein expression or cleavage.Our data, therefore, strongly indicate that side chain packing near the fusion peptide (Fig. ​(Fig.1A,1A, inset) is a strong modulator of Hendra virus F triggering, with a dramatic slowing in the rate of six-helix bundle formation observed when H372 is replaced with residues with smaller side chain volumes (Fig. ​(Fig.4).4). Modulation of side chain packing proximal to the FP could change the positioning of paramyxovirus FPs, thus altering the kinetics and efficiency of later conformational changes. Mutation of H372 may well stabilize interactions of the FP with the ectodomain following cleavage and thus affect triggering by substantially increasing the energy needed to project the FP toward the target cell membrane. Together, these data suggest a model by which packing around the fusion peptide affects both the rate and extent of F triggering.     

Structural features of paramyxovirus F protein required for fusion initiation

On the basis of the coordinates of the related Newcastle disease virus (NDV) F protein, Valine-94, a determinant of measles virus (MV) cytopathicity, is predicted to lie in a cylindrical cavity with 10 A diameter located at the F neck. A 16-residue domain around V94 is functionally interchangeable between NDV and MV F, supporting our homology model. Features of the cavity are conserved within the Paramyxovirinae. A hydrophobic base and a hydrophilic residue at the rim are required for surface expression. Small residue substitutions predicted to open the cavity were found to disrupt transport or limit fusogenicity of transport-competent mutants but can be compensated for by simultaneous insertion of larger residues at the opposing wall. Variants containing histidine substitutions mediate fusion at pH 8.5, while at pH 7.2 fusion is blocked, suggesting that functionality requires low charge in the cavity. These results indicate that specific structural features of the cavity are essential for paramyxovirus fusion initiation.     

A Stabilized Headless Measles Virus Attachment Protein Stalk Efficiently Triggers Membrane Fusion

Paramyxovirus attachment and fusion (F) envelope glycoprotein complexes mediate membrane fusion required for viral entry. The measles virus (MeV) attachment (H) protein stalk domain is thought to directly engage F for fusion promotion. However, past attempts to generate truncated, fusion-triggering-competent H-stem constructs remained fruitless. In this study, we addressed the problem by testing the hypothesis that truncated MeV H stalks may require stabilizing oligomerization tags to maintain intracellular transport competence and F-triggering activity. We engineered H-stems of different lengths with added 4-helix bundle tetramerization domains and demonstrate restored cell surface expression, efficient interaction with F, and fusion promotion activity of these constructs. The stability of the 4-helix bundle tags and the relative orientations of the helical wheels of H-stems and oligomerization tags govern the kinetics of fusion promotion, revealing a balance between H stalk conformational stability and F-triggering activity. Recombinant MeV particles expressing a bioactive H-stem construct in the place of full-length H are viable, albeit severely growth impaired. Overall, we demonstrate that the MeV H stalk represents the effector domain for MeV F triggering. Fusion promotion appears linked to the conformational flexibility of the stalk, which must be tightly regulated in viral particles to ensure efficient virus entry. While the pathways toward assembly of functional fusion complexes may differ among diverse members of the paramyxovirus family, central elements of the triggering machinery emerge as highly conserved.     

Mutations in the DI-DII Linker of Human Parainfluenza Virus Type 3 Fusion Protein Result in Diminished Fusion Activity

Human parainfluenza virus type 3 (HPIV3) can cause severe respiratory tract diseases in infants and young children, but no licensed vaccines or antiviral agents are currently available for treatment. Fusing the viral and target cell membranes is a prerequisite for its entry into host cells and is directly mediated by the fusion (F) protein. Although several domains of F are known to have important effects on regulating the membrane fusion activity, the roles of the DI-DII linker (residues 369–374) of the HPIV3 F protein in the fusogenicity still remains ill-defined. To facilitate our understanding of the role of this domain might play in F-induced cell-cell fusion, nine single mutations were engineered into this domain by site-directed mutagenesis. A vaccinia virus-T7 RNA polymerase transient expression system was employed to express the wild-type or mutated F proteins. These mutants were analyzed for membrane fusion activity, cell surface expression, and interaction between F and HN protein. Each of the mutated F proteins in this domain has a cell surface expression level similar to that of wild-type F. All of them resulted in a significant reduction in fusogenic activity in all steps of membrane fusion. Furthermore, all these fusion-deficient mutants reduced the amount of the HN-F complexes at the cell surface. Together, the results of our work suggest that this region has an important effect on the fusogenic activity of F.     

Potential electrostatic interactions in multiple regions affect human metapneumovirus f-mediated membrane fusion

The recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F(2) domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.     

Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H

Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex.Enveloped viruses gain access to target cells through fusion of viral and cellular membranes. This involves viral fusogenic envelope glycoprotein complexes that mediate membrane merger in a series of conformational rearrangements. For members of the Paramyxovirinae subfamily, fusion is accomplished by the concerted action of two glycoprotein complexes, the fusion (F) protein and the attachment protein (protein H, HN, or G, depending on the Paramyxovirinae genus) (21). Receptor binding by the attachment protein is thought to trigger refolding of the metastable F complex into the thermodynamically stable postfusion conformation and thus initiate the fusion event (20).Most paramyxoviruses require coexpression of homotypic envelope glycoproteins for efficient F triggering and membrane fusion (17, 45). This implicates specific protein-protein interactions between the F and the attachment protein in functional fusion complexes. All Paramyxovirinae attachment proteins form homo-oligomers. Their ectodomains are organized in a globular head domain that shows the six-blade propeller fold typical of sialidase structures (3, 5, 8, 15, 40, 49, 52) and a stalk domain connecting the head region to the transmembrane domain and short lumenal tail. Although no crystal information on the stalk domain is available, circular dichroism analyses of parainfluenza virus type 5 (PIV 5) HN (51) and structure predictions of measles virus (MeV) H and PIV 5 HN (22, 51) support an α-helical coiled-coil configuration of the stalk. Regions in the stalk have, furthermore, been implicated for several paramyxovirus HN proteins to mediate specificity for their homotypic F proteins (9, 10, 25, 45, 47). We have demonstrated that this extends to MeV H (22, 30), supporting the view that F-interacting residues may reside in the stalk region of the attachment protein (18, 30).Despite these advances, the effect of receptor binding on the attachment protein oligomer and the molecular mechanism linking receptor binding with F-protein refolding are poorly understood. MeV H head domains have been crystallized both free and complexed with soluble receptor in monomeric and dimeric forms (5, 15). Data available for attachment proteins of related Paramyxovirus family members, such as henipavirus G, and several HN proteins suggest that the tetramer (dimer of dimers) constitutes the physiological oligomer (2, 51, 52). By extension, this may equally apply to all attachment proteins of viruses of the Paramyxovirinae subfamily. Elucidating the oligomeric status of the attachment protein engaged in functional fusion complexes in situ will likely be paramount for the mechanistic understanding of paramyxovirus F triggering, given that no major conformational differences were observed between crystal structures of PIV 5 HN, human parainfluenza virus type 3 HN, henipavirus G, and MeV H solved alone or in complex with their receptor (3, 5, 8, 15, 40, 49, 52). It was, rather, hypothesized that receptor binding may affect the quaternary status of the attachment protein homo-oligomer, which could ultimately trigger F refolding (52). If a general theme applies to paramyxovirus entry, this brings into focus the question of whether a dimeric organization represents the physiological oligomer of native MeV H.Beyond the physical organization of the H oligomer, we have only begun to uncover some of the basic dynamics that govern the complex protein machineries required for membrane fusion and virus infection. For instance, it is unknown whether physical interaction of the attachment protein complex with F and induction of F triggering are separable events within an H oligomer, whether interaction of an H oligomer with multiple F complexes is required for optimal fusion activity, or even whether membrane fusion initiation requires engagement of every protein H monomer by the receptor.In the study described here, we have employed an integrated biochemical and functional approach to better understand some of the basic structural and mechanistic features of the MeV fusion complex. Blue native PAGE (BN-PAGE) analysis was used to test the organization of the native attachment protein oligomer extracted from purified viral particles and transiently transfected cells under different stringency conditions.Bimolecular protein complementation (BiC) allows the mechanistic assessment of multisubunit protein complexes. Application to the retrovirus envelope, for instance, has elucidated the interactions between HIV-1 gp120 and gp41 and the stoichiometry of the HIV-1 envelope trimer during entry (39, 50). By applying this concept to the paramyxovirus glycoprotein system, we have newly developed an H BiC (H-BiC) assay for MeV protein H that complements BN-PAGE data with functional information and probes the stoichiometric requirements for the organization of biologically active fusion complexes and the receptor-mediated initiation of fusion. Results are interpreted in light of current hypotheses regarding the possible effects of receptor binding on paramyxovirus attachment proteins.     

Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy.     

Mutations in the cytoplasmic domain of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion

SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.     

Directional Information Transfer in Protein Helices

IN the course of studies on the relation between conformation and primary sequence in globular proteins, it has become clear that the choice a residue makes between a right handed α-helical conformation (H?) and any other conformation (H) is determined mainly by that residue and its near neighbours in the primary sequence^1–9. When looking for physical mechanisms to explain these findings it is important to know whether the influence of one residue on the conformational state of a neighbouring residue has any directional characteristics. The possibility of certain residues exerting helix-forming influence in either the COOH-terminal or NH₂-terminal direction preferentially is suggested by the non-random distribution of amino-acid residues between the two ends of helical regions in globular proteins^4,10. Ptitsyn's analysis⁴ suggested that a group of residues containing alanine and leucine tends to occur within helical regions, while positively and negatively charged side chain residues are distributed preferentially at the carboxyl-and amino-terminal ends, respectively, of helices.