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.     

Characterization of a Natural Mutation in an Antigenic Site on the Fusion Protein of Measles Virus That Is Involved in Neutralization

Although measles virus is an antigenically monotypic virus, nucleotide sequence analysis of the hemagglutinin and nucleoprotein genes has permitted the differentiation of a number of genotypes. In contrast, the fusion (F) protein is highly conserved; only three amino acid changes have been reported over a 40-year period. We have isolated a measles virus strain which did not react with an anti-F monoclonal antibody (MAb) which we had previously shown to be directed against a dominant antigenic site. This virus strain, Lys-1, had seven amino acid changes compared with the Edmonston strain. We have shown that a single amino acid at position 73 is responsible for its nonreactivity with the anti-F MAb. With the same MAb, antibody-resistant mutants were prepared from the vaccine strain. A single amino acid change at position 73 (R→W) was observed. The possibility of selecting measles virus variants in vaccinated populations is discussed.     

副粘病毒附着蛋白在病毒融合过程中的作用

副粘病毒附着蛋白（AP）是病毒表面的一种主要糖蛋白,它能够诱导机体产生中和抗体。近年来的研究表明附着蛋白在病毒融合过程中的作用不公限于其对受体的识别和结合,它还促进融合蛋白（F）介导病毒与宿主细胞的融合。由此可见,副粘病毒具有其特有的融合机理,因此研究附着蛋白在病毒融合过程中的作用是揭示副粘病毒融合机理的前提,同时也会为新型抑制药物的研究提供思路。     

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.     

Reversible Inhibition of Fusion Activity of a Paramyxovirus Fusion Protein by an Engineered Disulfide Bond in the Membrane-Proximal External Region

Cysteines were introduced into the membrane-proximal external region (MPER) of the paramyxovirus F protein. A disulfide bond formed, and the mutant protein was expressed at the cell surface but was fusion inactive. Reduction of the disulfide bond restored fusion activity. The data indicate that in addition to dissociation of the three-helix bundle stalk domain of prefusion F, the MPER region also needs to separate for F to be able to refold and cause fusion.     

Regulation of Paramyxovirus Fusion Activation: the Hemagglutinin-Neuraminidase Protein Stabilizes the Fusion Protein in a Pretriggered State

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.     

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.     

Functional Analysis of the Transmembrane Domain in Paramyxovirus F Protein-Mediated Membrane Fusion

To enter cells, enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single-cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the paramyxovirus parainfluenza virus 5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion.     

Membrane Fusion Promoted by Increasing Surface Densities of the Paramyxovirus F and HN Proteins: Comparison of Fusion Reactions Mediated by Simian Virus 5 F, Human Parainfluenza Virus Type 3 F, and Influenza Virus HA

The membrane fusion reaction promoted by the paramyxovirus simian virus 5 (SV5) and human parainfluenza virus type 3 (HPIV-3) fusion (F) proteins and hemagglutinin-neuraminidase (HN) proteins was characterized when the surface densities of F and HN were varied. Using a quantitative content mixing assay, it was found that the extent of SV5 F-mediated fusion was dependent on the surface density of the SV5 F protein but independent of the density of SV5 HN protein, indicating that HN serves only a binding function in the reaction. However, the extent of HPIV-3 F protein promoted fusion reaction was found to be dependent on surface density of HPIV-3 HN protein, suggesting that the HPIV-3 HN protein is a direct participant in the fusion reaction. Analysis of the kinetics of lipid mixing demonstrated that both initial rates and final extents of fusion increased with rising SV5 F protein surface densities, suggesting that multiple fusion pores can be active during SV5 F protein-promoted membrane fusion. Initial rates and extent of lipid mixing were also found to increase with increasing influenza virus hemagglutinin protein surface density, suggesting parallels between the mechanism of fusion promoted by these two viral fusion proteins.     

副粘病毒融合蛋白活性位点中亮氨酸基因突变分析

为了确定副粘病毒融合蛋白（Ｆ）分子上活性位点中亮氨酸在Ｆ的细胞融合作用中的作用,弄清Ｆ融合细胞的分子机理,采用基因定点突变法创造一个酶切位点,用酶切反应初步筛选突变株,然后用ＤＮＡ序列分析进一步确定,并在真核细胞内进行表达,Ｇｉｅｍｓａ染色和指示基因法检测细胞融合功能,荧光强度分析（ＦＡＣＳ）检测表达效率。结果表明,ｈＰＩＶ３等４６０位亮氨酸（Ｌ）和第４７４位异亮氨酸（Ｉ）分别突变成丙氨酸（Ａ）（     

新城疫病毒ZJ1株F蛋白裂解位点突变对其融合活性的影响

新城疫病毒ZJ1毒株是近年来在我国水禽中流行并能引起水禽严重发病和死亡的强毒株,其F蛋白裂解位点有多个碱性氨基酸分布。将该毒株F蛋白裂解位点的112、115和117位碱性氨基酸突变成弱毒株特征的非碱性氨基酸,构建了重组表达质粒pCI-FT。分别将突变前后的F蛋白与该毒株的HN蛋白在COS-1细胞共表达,表明突变前后的F蛋白均有融合活性;分别将突变前后的F蛋白与该毒株的HN蛋白在CEF细胞共表达,表明突变后F蛋白被裂解的活性大大降低。以上研究为下一步在全长cDNA克隆水平上对F蛋白裂解位点氨基酸序列进行相应突变,研究毒力相关因素以及构建毒力致弱疫苗株等奠定基础。     

Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity

Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.     

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.     

Compensatory Point Mutations in the Human Immunodeficiency Virus Type 1 Gag Region That Are Distal from Deletion Mutations in the Dimerization Initiation Site Can Restore Viral Replication

The dimerization initiation site (DIS), downstream of the long terminal repeat within the human immunodeficiency virus type 1 (HIV-1) genome, can form a stem-loop structure (SL1) that has been shown to be involved in the packaging of viral RNA. In order to further determine the role of this region in the virus life cycle, we deleted the 16 nucleotides (nt) at positions +238 to +253 within SL1 to generate a construct termed BH10-LD3 and showed that this virus was impaired in viral RNA packaging, viral gene expression, and viral replication. Long-term culture of these mutated viruses in MT-2 cells, i.e., 18 passages, yielded revertant viruses that possessed infectivities similar to that of the wild type. Cloning and sequencing showed that these viruses retained the original 16-nt deletion but possessed two additional point mutations, which were located within the p2 and NC regions of the Gag coding region, respectively, and which were therefore named MP2 and MNC. Site-directed mutagenesis studies revealed that both of these point mutations were necessary to compensate for the 16-nt deletion in BH10-LD3. A construct with both the 16-nt deletion and the MP2 mutation, i.e., LD3-MP2, produced approximately five times more viral protein than BH10-LD3, while the MNC mutation, i.e., construct LD3-MNC, reversed the defects in viral RNA packaging. We also deleted nt +261 to +274 within the 3′ end of SL1 and showed that the diminished infectivity of the mutated virus, termed BH10-LD4, could also be restored by the MP2 and MNC point mutations. Therefore, compensatory mutations within the p2 and NC proteins, distal from deletions within the DIS region of the HIV genome, can restore HIV replication, viral gene expression, and viral RNA packaging to control levels.     

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.