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.     

Critical Role of the Fusion Protein Cytoplasmic Tail Sequence in Parainfluenza Virus Assembly

Interactions between viral glycoproteins, matrix protein and nucleocapsid sustain assembly of parainfluenza viruses at the plasma membrane. Although the protein interactions required for virion formation are considered to be highly specific, virions lacking envelope glycoprotein(s) can be produced, thus the molecular interactions driving viral assembly and production are still unclear. Sendai virus (SeV) and human parainfluenza virus type 1 (hPIV1) are highly similar in structure, however, the cytoplasmic tail sequences of the envelope glycoproteins (HN and F) are relatively less conserved. To unveil the specific role of the envelope glycoproteins in viral assembly, we created chimeric SeVs whose HN (rSeVhHN) or HN and F (rSeVh(HN+F)) were replaced with those of hPIV1. rSeVhHN grew as efficiently as wt SeV or hPIV1, suggesting that the sequence difference in HN does not have a significant impact on SeV replication and virion production. In sharp contrast, the growth of rSeVh(HN+F) was significantly impaired compared to rSeVhHN. rSeVh(HN+Fstail) which expresses a chimeric hPIV1 F with the SeV cytoplasmic tail sequence grew similar to wt SeV or rSeVhHN. Further analysis indicated that the F cytoplasmic tail plays a critical role in cell surface expression/accumulation of HN and F, as well as NP and M association at the plasma membrane. Trafficking of nucelocapsids in infected cells was not significantly affected by the origin of F, suggesting that F cytoplasmic tail is not involved in intracellular movement. These results demonstrate the role of the F cytoplasmic tail in accumulation of structural components at the plasma membrane assembly sites.     

Full Conversion of the Hemagglutinin-Neuraminidase Specificity of the Parainfluenza Virus 5 Fusion Protein by Replacement of 21 Amino Acids in Its Head Region with Those of the Simian Virus 41 Fusion Protein

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.     

Parainfluenza Virus 5 Expressing the G Protein of Rabies Virus Protects Mice after Rabies Virus Infection

Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) strain expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection.     

Mutations in the Putative Dimer-Dimer Interfaces of the Measles Virus Hemagglutinin Head Domain Affect Membrane Fusion Triggering

Measles virus (MV), an enveloped RNA virus belonging to the Paramyxoviridae family, enters the cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin (H) and a fusion (F) protein. The crystal structure of the receptor-binding head domain of MV-H bound to its cellular receptor revealed that the MV-H head domain forms a tetrameric assembly (dimer of dimers), which occurs in two forms (forms I and II). In this study, we show that mutations in the putative dimer-dimer interface of the head domain in either form inhibit the ability of MV-H to support membrane fusion, without greatly affecting its cell surface expression, receptor binding, and interaction with the F protein. Notably, some anti-MV-H neutralizing monoclonal antibodies are directed to the region around the dimer-dimer interface in form I rather than receptor-binding sites. These observations suggest that the dimer-dimer interactions of the MV-H head domain, especially that in form I, contribute to triggering membrane fusion, and that conformational shift of head domain tetramers plays a role in the process. Furthermore, our results indicate that although the stalk and transmembrane regions may be mainly responsible for the tetramer formation of MV-H, the head domain alone can form tetramers, albeit at a low efficiency.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

Insertional Mutations in Herpes Simplex Virus Type 1 gL Identify Functional Domains for Association with gH and for Membrane Fusion

Glycoprotein L (gL) is one of four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion. This glycoprotein oligomerizes with gH to form a membrane-bound heterodimer but can be secreted when expressed without gH. Twelve unique gL linker-insertion mutants were generated to identify regions critical for gH binding and gH/gL processing and regions essential for cell fusion and viral entry. All gL mutants were detected on the cell surface in the absence of gH, suggesting incomplete cleavage of the signal peptide or the presence of a cell surface receptor for secreted gL. Coexpression with gH enhanced the levels of cell surface gL detected by antibodies for all gL mutants except those that were defective in their interactions with gH. Two insertions into a conserved region of gL abrogated the binding of gL to gH and prevented gH expression on the cell surface. Three other insertions reduced the cell surface expression of gH and/or altered the properties of gH/gL heterodimers. Altered or absent interaction of gL with gH was correlated with reduced or absent cell fusion activity and impaired complementation of virion infectivity. These results identify a conserved domain of gL that is critical for its binding to gH and two noncontiguous regions of gL, one of which contains the conserved domain, that are critical for the gH/gL complex to perform its role in membrane fusion.Glycoprotein L (gL) is one of the four glycoproteins required for the entry of herpes simplex virus (HSV) into cells and for virus-induced cell fusion (26, 33). The others are gB, gD, and gH (30). The functional unit containing gL is a heterodimer formed with gH (gH/gL) (15). Because mature gL has no membrane-spanning domain, other than a cleavable signal peptide, it is secreted unless it is coexpressed with gH, a type 1 glycoprotein that anchors gL to the cell membrane (2). Also, gH is not properly processed or transported out of the endoplasmic reticulum unless it is coexpressed with gL (15).Most, if not all, herpesviruses express orthologs of gB, gH, and gL, which are believed to form the core membrane-fusing machinery necessary for viral entry and cell fusion. For some herpesviruses, such as Epstein-Barr virus and human cytomegalovirus, the gH/gL oligomer may contain additional viral subunits that can influence binding of the complex to cell receptors and determine cell tropism (14, 34, 35). For HSV, however, only gD and gB have been shown to have receptor-binding activities that are required for entry (27, 31). Although HSV gH has an RGD motif and the gH/gL heterodimer can bind to certain integrins, this binding seems not to be necessary for viral entry (3, 22).The initial interaction of HSV with cells can be the reversible attachment of virus to cell surface heparan sulfate, mediated by viral glycoprotein gB and/or gC (29). Then, gD can bind to one of its receptors, including herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor receptor family; nectin-1 or nectin-2, cell adhesion molecules belonging to the immunoglobulin superfamily; or specific sites in heparan sulfate generated by 3-O-sulfotransferases (31). In addition to binding to heparan sulfate, gB can also bind to other cell surface receptors, including paired immunoglobulin-like receptor alpha (PILRα) (27). Binding of both gD and gB to one of their respective receptors appears to be required for triggering the membrane-fusing activity of gB and/or gH/gL, which leads to viral entry.A recent X-ray structure of HSV type 1 (HSV-1) gB suggests that it is a class III viral fusogen similar in domain organization, but not primary sequence, to the G protein of vesicular stomatitis virus (13). It has been proposed that HSV-1 gH has features characteristic of class I viral fusogens, such as putative heptad repeats and fusion peptides (6, 9-11). Also, peptides matching the sequence of gH can interact with lipids and/or induce the fusion of lipid vesicles (4, 5, 8). Hemifusion between cells and between virus and cell can be induced by gH/gL and gD in the absence of gB (32). Many questions remain about the respective roles of gH/gL and gB in inducing membrane fusion.The four conserved cysteines in gL were found to be essential for gL-gH association and function (1). Mutational analyses of gL by C-terminal deletions showed that the first 147 amino acids of gL are sufficient for association with gH but that the first 161 amino acids are necessary for cotransport of gH and gL to the cell surface (17, 23) and for gL activity in cell fusion and viral entry (17). Lastly, certain anti-gL monoclonal antibodies (MAbs) can inhibit cell fusion but not viral entry, despite demonstrable binding of the MAbs to virus, suggesting that gL may play a different role in each process (21). These MAbs were mapped to the C-terminal region of gL (21, 23). The diagram at the bottom of Fig. ​Fig.11 shows the locations of the gL features mentioned above and of the signal peptide.Open in a separate windowFIG. 1.Effects of insertional mutations on HSV-1 gL and gH cell surface expression. CHO cells were transfected with plasmids expressing gH and WT gL or a gL mutant. Cell surface expression of gL and gH was quantified by CELISA using polyclonal R88 antiserum (filled circles) and MAb 52S (open triangles), respectively. A linear representation of the gL polypeptide is shown below the graph, with coded bars identifying features of gL. The bars represent the signal peptide (uncolored hatched), the N-terminal 161-amino-acid fragment necessary for the formation of functional gH/gL complexes (dark gray), highly conserved residues within this fragment (cross-hatched dark- gray bar), and epitopes recognized by a panel of anti-gL MAbs (light-gray and uncolored vertically striped bars). The values presented for cell surface expression of each mutant gL and of cotransfected WT gH are means from three independent experiments expressed as percentages of WT gL (or of gH cotransfected with WT gL) values, after subtraction of background values obtained in the absence of gL expression and as a function of the position of the insertion. Standard deviations are presented in Fig. ​Fig.22 and ​and33 for similar experiments.The interactions between gL and gH required for proper intracellular transport, processing, and cell surface expression make it difficult to investigate the functional role of one of these glycoproteins in cell fusion and viral entry independently of the other. We generated a panel of gL linker-insertion mutants to identify regions critical for gH binding and transport and regions essential for cell fusion and viral entry. One aim was to determine whether these roles of gL could be dissociated or were linked. Characterization of 12 unique gL linker-insertion mutants showed that (i) a conserved domain of gL is critical for the physical interaction of gL with gH and for the normal processing of gH, (ii) two noncontiguous regions of gL, one of which contains the highly conserved domain, are critical for the normal conformation and function of gH/gL heterodimers, and (iii) wild-type (WT) and mutant gLs can be detected on the cell surface in the absence of gH, suggesting the possibility of an independent role for uncomplexed gL. These results support and extend previous studies suggesting that gL has a larger role in membrane fusion than serving as a chaperone for gH and that specific mutations in gL can influence the function of the gH/gL heterodimer.     

Mutations in the Ectodomain of Newcastle Disease Virus Fusion Protein Confer a Hemagglutinin-Neuraminidase-Independent Phenotype

The entry of enveloped viruses into host cells is preceded by membrane fusion, which in paramyxoviruses is triggered by the fusion (F) protein. Refolding of the F protein from a metastable conformation to a highly stable postfusion form is critical for the promotion of fusion, although the mechanism is still not well understood. Here we examined the effects of mutations of individual residues of the F protein of Newcastle disease virus, located at critical regions of the protein, such as the C terminus of the N-terminal heptad repeat (HRA) and the N terminus of the C-terminal heptad repeat (HRB). Seven of the mutants were expressed at the cell surface, showing differences in antibody reactivity in comparison with the F wild type. The N211A, L461A, I463A, and I463F mutants showed a hyperfusogenic phenotype both in syncytium and in dye transfer assays. The four mutants promoted fusion more efficiently at lower temperatures than the wild type did, meaning they probably had lower energy requirements for activation. Moreover, the N211A, I463A, and I463F mutants exhibited hemagglutinin-neuraminidase (HN)-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.Newcastle disease virus (NDV) is an avian enveloped virus belonging to the family Paramyxoviridae. Two viral membrane-associated proteins are responsible for the entry of the virus into the host cell: they are hemagglutinin-neuraminidase (HN), a receptor-binding protein that interacts with sialoglycoconjugates at the cell surface, and F, a trimeric class I fusion protein that, upon activation, triggers the fusion of the viral and target membranes. F protein is activated after the attachment of its homotypic HN protein to the proper receptor; however, how HN activates F is not well understood. F protein is synthesized as an inactive precursor, F₀, that is activated by proteolytic cleavage to the disulfide-linked F₁-F₂ fusion-competent form (Fig. ​(Fig.1)1) (10). The crystal structures of several paramyxoviral fusion proteins, in both the prefusion and postfusion conformations (3, 26, 27), have revealed that these proteins undergo major conformational changes, from a metastable conformation to a highly stable, postfusion form. Several regions in the ectodomain of class I viral fusion proteins are involved in these conformational conversions, including a hydrophobic fusion peptide at the N terminus of the F1 protein and two hydrophobic heptad repeat motifs, HRA and HRB, located at its N and C termini, respectively (Fig. ​(Fig.1).1). In the prefusion form, HRB shows a triple-stranded coiled-coil conformation forming the stalk of the mushroom-like protein (3, 19, 27). Its globular head contains three domains, DI to DIII (Fig. ​(Fig.1),1), with the base of the head being formed by the DI and DII domains, with residues predominantly located between HRA and HRB. The top of the head is formed by DIII, consisting mainly of HRA and the fusion peptide, located on the side of the head sequestered between adjacent subunits. In this prefusion state, HRA is folded as two antiparallel β-strands and four (h1 to h4) helices (27) (see Fig. ​Fig.6).6). The DIII domain undergoes major structural changes from the prefusion to the final postfusion conformation. HRA refolds as an α-helix, propelling the fusion peptide into the target membrane and generating a prehairpin intermediate (see Fig. ​Fig.6).6). The final, stable conformation consists of a six-helical bundle (6HB), comprising a dimer of trimers in which the trimeric HRA coiled coil forms the core, packed along the outside by three antiparallel HRB α-helices (1, 3, 19, 27).Open in a separate windowFIG. 1.Schematic representation of the structure of the NDV fusion protein. (A) Domain structure of F protein (27). (B) Locations of the fusion peptide, HR regions, and sequences studied. Mutated residues are indicated in bold.Open in a separate windowFIG. 6.Scheme of conformational changes in HRA from prefusion to postfusion state. (A) Ribbon model of PIV5 F protein in its metastable prefusion conformation (PDB accession number 2b9b) (27), showing some residues (named in white) from the A subunit and the corresponding residues in the NDV F protein (named in yellow). Subunits B and C are depicted in gray for clarity. (B) In the metastable, prefusion conformation, HRA is folded as a spring-loaded mixture of α-helices, turns, and β-strands, comprising 11 segments in the DIII head domain of the trimer (27). (C) After fusion, HRA is presented as a single long helix that allows the fusion peptide to be buried in the target membrane. The approximate positions of HRC and the core β-sheet are shown as dashed lines for both conformations.The refolding mechanism that triggers F protein activation is still not well understood. Mutational analysis of the HRA and HRB domains of paramyxovirus F proteins (3, 13, 18, 19, 22, 23), as well as the use of HRA- and HRB-derived peptides (6, 17), has led to the proposal of a series of discrete refolding intermediates of the F protein, from the metastable native conformation, through the prehairpin intermediate, and to the final postfusion hairpin structure (6HB) (17, 19, 27). To gain further insight into the individual residues critical for this mechanism, in this work we mutated several residues of the head and stalk of the NDV F protein (Fig. ​(Fig.1).1). The mutations disrupted F protein antibody reactivity, fusogenicity, and HN dependence in different ways. Interestingly, a mutant of the C-terminal h4 α-helix of HRA (N211A mutant) and two mutants of a residue located at the most N-terminal position of HRB (I463A and I463F mutants) exhibited a hyperfusogenic phenotype and HN-independent activity when influenza virus hemagglutinin (HA) was coexpressed as an attachment protein. The data are discussed in terms of alterations of the refolding pathway and/or the stability of the prefusion and fusion conformations.     

Human Parainfluenza Virus Infection of the Airway Epithelium: Viral Hemagglutinin-Neuraminidase Regulates Fusion Protein Activation and Modulates Infectivity

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN′s receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN′s F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN′s F-triggering activity may have promise in vivo.Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry, the viral surface glycoproteins hemagglutinin-neuraminidase (HN) (the receptor-binding molecule) and F (the fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. To understand these mechanisms, elucidate how paramyxoviruses enter cells, and develop strategies to prevent or treat infection, we study human parainfluenza virus (HPIV), an important cause of croup and bronchiolitis in children. Our results have uncovered fundamental roles of the receptor-binding protein in paramyxovirus fusion and principles of coordinated interaction between the glycoproteins during the viral life cycle.To understand how the diverse functions of the viral glycoproteins are regulated during the viral life cycle, we have used viruses bearing variant HN molecules with mutations at the binding/F-triggering site (and/or the primary receptor-binding site) to study how this molecule works to trigger F (2, 3, 7, 10, 15, 18, 20). The correct timing of F activation (triggering) by HN is essential for entry. For infection to occur, triggering must occur only when F is in proximity to the target cell membrane, and we propose that the regulation of F triggering is essential for the survival of the virus. The outcome of infection is determined by the target cell''s properties and its receptors, and specific mechanisms that are relevant to pathogenesis need to be tested using tissues that reflect the natural host. We therefore tested the hypothesis that a dysregulation of F triggering precludes successful infection in both a cotton rat model and the natural host airway epithelium.For the cotton rat model, previous studies suggested that altered pathogenesis in HPIV infection might be caused by specific HN mutations (24). The present detailed studies of the cotton rat using HN viral variants suggest that the extent of lung infection correlates with the ability of each variant to grow in vivo. The most striking finding is that the ability of the HN variants to grow in vivo is inversely related to their ability to fuse a monolayer of cultured cells. In order to understand the determinants of infection in the natural host, we therefore turned to a model that closely reflects the natural human host tissue, the human airway epithelium (HAE). This model utilizes a recently developed method for culturing primary HAE cells at an air-liquid interface, generating a differentiated, pseudostratified, mucociliary epithelium that faithfully represents the HAE (16). The HAE model was previously used to characterize the polarity and cell specificity of respiratory syncytial virus (26) and HPIV type 3 (HPIV3) (25), confirming that it is suited to studying paramyxovirus-HAE interactions that reflect those in the human lung.We used viruses bearing HNs that are altered in receptor binding or F triggering to reveal the functional relevance of these properties in the HAE and to establish the key role of HN binding site II in infection in the natural host. We propose that an enhanced triggering of F by HN may be a disadvantage in vivo and that the function and timing of F triggering are critical in the target tissue. The correct balance between the three functions of HN (receptor binding, receptor cleaving, and F triggering) resides in the functions of the two binding sites (18), binding and release in site I and F triggering in site II, and both sites of HN play key roles in the natural host.     

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.     

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.     

On the Stability of Parainfluenza Virus 5 F Proteins

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.     

Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.     

Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro

RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNA_i^Met. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNA_i^Met turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNA_i^Met turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.