Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation.

Enveloped viruses have evolved complex glycoprotein machinery that drives the fusion of viral and cellular membranes, permitting entry of the viral genome into the cell. For the paramyxoviruses, the fusion (F) protein catalyses this membrane merger and entry step, and it has been postulated that the F protein undergoes complex refolding during this process. Here we report the crystal structure of the parainfluenza virus 5 F protein in its prefusion conformation, stabilized by the addition of a carboxy-terminal trimerization domain. The structure of the F protein shows that there are profound conformational differences between the pre- and postfusion states, involving transformations in secondary and tertiary structure. The positions and structural transitions of key parts of the fusion machinery, including the hydrophobic fusion peptide and two helical heptad repeat regions, clarify the mechanism of membrane fusion mediated by the F protein.

Detailed genetic analyses of the HN gene in human respirovirus 3 detected in children with acute respiratory illness in the Iwate Prefecture, Japan.

We performed detailed genetic analyses of the partial hemagglutinin-neuraminidase (HN) gene in 34 human respirovirus 3 (HRV3) strains from children with acute respiratory illness during 2013-2015 in Iwate Prefecture, Japan. In addition, we performed analyses of the evolutionary timescale of the gene using the Bayesian Markov chain Monte Carlo (MCMC) method. Furthermore, we analyzed pairwise distances and performed selective pressure analyses followed by linear B-cell epitope mapping and N-glycosylation and phylodynamic analyses. A phylogenetic tree showed that the strains diversified at around 1939, and the rate of molecular evolution was 7.6 × 10-4 substitutions/site/year. Although the pairwise distances were relatively short (0.03 ±â€¯0.018 [mean ±â€¯standard deviation, SD]), two positive selection sites (Cys544Trp and Leu555Ser) and no amino acid substitutions were found in the active/catalytic sites. Six epitopes were estimated in this study, and three mouse monoclonal antibody binding sites (amino acid positions 278, 281, and 461) overlapped with two epitopes belonging to subcluster C3 strains. Bayesian skyline plot analyses indicated that subcluster C3 strains have been increasing from 2004, whereas subcluster C1 strains have declined from 2004. Based on these results, Iwate strains were divided into two subclusters and each subcluster evolved independently. Moreover, our results suggested that some predicted linear epitopes (epitopes 3 and 5) are candidates for an HRV3 vaccine motif. To better understand the details of the molecular evolution of HRV, further studies are needed.

The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway.

Fusion of Sendai virus and individual host cells and inhibition of fusion by lipophosphoglycan measured with image correlation spectroscopy.

Fusion between Sendai virus (SV) and individual host cells was investigated with confocal laser scanning microscopy (CLSM) and image correlation spectroscopy (ICS). SV was labeled with the fluorescent probe 7-octadecylamino-4-nitrobenz-2-oxa-1,3-diazole (NBD-NH-C18) and was allowed to bind to host cells (HEp-2, BALB-3T3) at 4 degrees C. The effect of lipophosphoglycan (LPG), isolated from Leishmania donovani, on virus fusion was investigated by incorporation of LPG (0, 5, 10 or 20 microM) into the host cell membrane (HEp-2) before addition of SV. LPG did not affect the number of SV bound per cell. After incubation at 37 degrees C for 15 min without LPG, CLSM revealed a redistribution of NBD-NH-C18 from the SV envelope to the host cell membrane and an increase in average fluorescence intensity, indicating dequenching. ICS analysis of images obtained after incubation at 37 degrees C showed an increased mean cluster density to 260% of the value at 4 degrees C, reflecting the disappearance of labeled SV from the cell surface and diffusion of NBD-NH-C18 into the host cell membrane. Preincubation of the cells with LPG inhibited the temperature-induced redistribution and dequenching of NBD-NH-C18 in a concentration-dependent manner, with a total inhibition of fusion at 20 microM LPG. Together, the results demonstrate that CLSM combined with ICS is a powerful tool for studies of fusion of enveloped viruses with individual host cells and that LPG inhibits the fusion process at or before the hemifusion (lipid mixing) stage of SV interaction with cells.

LearnCoil-VMF: computational evidence for coiled-coil-like motifs in many viral membrane-fusion proteins.

Crystallographic studies have shown that the coiled-coil motif occurs in several viral membrane-fusion proteins, including HIV-1 gp41 and influenza virus hemagglutinin. Here, the LearnCoil-VMF program was designed as a specialized program for identifying coiled-coil-like regions in viral membrane-fusion proteins. Based upon the use of LearnCoil-VMF, as well as other computational tools, we report detailed sequence analyses of coiled-coil-like regions in retrovirus, paramyxovirus and filovirus membrane-fusion proteins. Additionally, sequence analyses of these proteins outside their putative coiled-coil domains illustrate some structural differences between them. Complementing previous crystallographic studies, the coiled-coil-like regions detected by LearnCoil-VMF provide further evidence that the three-stranded coiled coil is a common motif found in many diverse viral membrane-fusion proteins. The abundance and structural conservation of this motif, even in the absence of sequence homology, suggests that it is critical for viral-cellular membrane fusion. The LearnCoil-VMF program is available at http://web.wi.mit.edu/kim

Paramyxovirus F1 protein has two fusion peptides: implications for the mechanism of membrane fusion.

Viral fusion proteins contain a highly hydrophobic segment, named the fusion peptide, which is thought to be responsible for the merging of the cellular and viral membranes. Paramyxoviruses are believed to contain a single fusion peptide at the N terminus of the F1 protein. However, here we identified an additional internal segment in the Sendai virus F1 protein (amino acids 214-226) highly homologous to the fusion peptides of HIV-1 and RSV. A synthetic peptide, which includes this region, was found to induce membrane fusion of large unilamellar vesicles, at concentrations where the known N-terminal fusion peptide is not effective. A scrambled peptide as well as several peptides from other regions of the F1 protein, which strongly bind to membranes, are not fusogenic. The functional and structural characterization of this active segment suggest that the F1 protein has an additional internal fusion peptide that could participate in the actual fusion event. The presence of homologous regions in other members of the same family suggests that the concerted action of two fusion peptides, one N-terminal and the other internal, is a general feature of paramyxoviruses.

The roles of individual cysteine residues of Sendai virus fusion protein in intracellular transport.

The role of intramolecular disulfide bonds in the fusion (F) protein of Sendai virus was studied. The 10 cysteine residues were changed to serine residues using site-directed mutagenesis. None of the cysteine mutant F proteins reacted with a monoclonal antibody specific for the mature conformation of the F protein, but eight of ten mutants reacted with an immature conformation-specific monoclonal antibody. The transport of these mutant proteins to the cell surface was drastically reduced. All of the cysteine mutant F proteins remained sensitive to endoglycosidase H (endo H) for 3 h after their synthesis. Moreover, cell surface transport of the hemagglutinin-neuraminidase (HN) protein co-expressed with each of these cysteine mutant F proteins was also reduced. These results suggest that all cysteine residues participate in the formation of intramolecular disulfide bonds, that co-translational disulfide bond formation is crucial to the correct folding and intracellular transport of the F protein, and that interaction of the F and HN proteins takes place intracellulary.

Functional analysis of the individual oligosaccharide chains of sendai virus fusion protein.

The roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking g1 and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.

Efficient transfer of intact oligonucleotides into the nucleus of ligament scar fibroblasts by HVJ-cationic liposomes is correlated with effective antisense gene inhibition.

The efficacy of two different cationic liposomes, Lipofectin and hemagglutinating virus of Japan (HVJ)-cationic liposomes, on nuclear uptake of fluorescence-labeled phosphorothioate oligodeoxyribonucleotide (S-ODN) by ligament scar fibroblasts and suppression of decorin mRNA expression when antisense decorin S-ODN was transferred was investigated. There was no significant difference in nuclear uptake of fluorescent ODN between the two methods. However, only HVJ-cationic liposomes had a significant effect on suppression of decorin mRNA expression levels. To address the discrepancy, the molecular integrity of the transferred ODN in the cells was assessed by analysis of fluorescence resonance energy transfer (FRET) within double-fluorescence-labeled S-ODN. More than 70% of the ODN transfected by HVJ-cationic liposomes remained intact within the nucleus at 20 h after transfection, while the majority of the ODN transferred by Lipofectin was degraded at this point. These results suggest a strong relationship between the nuclear integrity of transfected antisense ODN and its suppression of target mRNA expression.

Membrane structure of the hepatitis B virus surface antigen particle.

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.

The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.

Characterization of a Paramyxovirus from a Fer de Lance viper (Bothrops jararaca): partial nucleotide sequence of the putative fusion protein.

During the generation of Expressed Sequence Tags (ESTs) from the Fer de Lance viper (Bothrops jararaca) venom glands, a partial cDNA (clone H8) coding for a protein with all the features of a paramyxovirus fusion protein was characterized. It has 920 bp and codes for a partial protein of 279 amino acids. Two potential N-glycosylation sites are present in the sequence which also possesses a typical membrane anchoring domain made of a stretch of hydrophobic amino acids. The polyadenylation signal sequence was identified. When compared to other fusion proteins, it showed the highest sequence similarity (37-39%) with those of human parainfluenza 3 and Sendai virus.

Tetrameric coiled coil domain of Sendai virus phosphoprotein.

The high resolution X-ray structure of the Sendai virus oligomerization domain reveals a homotetrameric coiled coil structure with many details that are different from classic coiled coils with canonical hydrophobic heptad repeats. Alternatives to the classic knobs-into-holes packing lead to differences in supercoil pitch and diameter that allow water molecules inside the core. This open and more hydrophilic structure does not seem to be destabilized by mutations that would be expected to disrupt classic coiled coils.

Studies with cross-linking reagents on the oligomeric form of the paramyxovirus fusion protein.

The oligomeric form of the paramyxovirus simian virus 5 (SV5) fusion (F) glycoprotein has been examined by using chemical cross-linking and sucrose density gradient fractionation. In addition, chemical cross-linking was used to examine the kinetics of assembly of the F oligomer. Analysis by SDS-PAGE on 3.5% gels of the cross-linked F molecules indicated three major species with calculated molecular weights of M(r) approximately 65, M(r) approximately 130, and M(r) approximately 195 kDa, suggesting F monomers, dimers, and trimers, respectively. The cross-linked F species of M(r) approximately 195 kDa migrated on gels faster than influenza virus hemagglutinin trimers and between the dimeric and tetrameric forms of paramyxovirus hemagglutinin-neuraminidase (HN). Furthermore, the F protein oligomer was found to sediment slower than the HN tetramer on sucrose gradient centrifugation. SDS-PAGE analysis of the cross-linked F protein of Newcastle disease virus and human parainfluenza virus 3 showed a pattern very similar to that found for SV5. The data are consistent with those expected for the paramyxovirus F protein being a homotrimer.

Multiple amino acid substitutions in the HN protein of the paramyxovirus, SV5, are selected for in monoclonal antibody resistant mutants.

Monoclonal antibody resistant (MAR) mutants (which escaped antibody-mediated neutralization) were selected from simian (W 3) and human (LN) isolates of simian virus 5 (SV 5), using monoclonal antibodies (MAbs) specific for antigenic sites 4 and 5 on the HN glycoprotein. Resistance correlated with an inability of the selecting antibody to bind with the respective MAR mutants. Sequence comparisons between parental and mutant HN proteins revealed multiple non-adjacent amino acid substitutions in the majority of MAR mutants. The same multiple substitutions were identified in mutants selected from both the LN and W 3 isolates of SV 5. Furthermore, different mutations on the primary sequence of the HN protein conferred resistance to the same MAb.

Sequence analyses of human parainfluenza virus type 4A and type 4B fusion proteins.

cDNAs encoding human parainfluenza virus type 4A and type 4B (hPIV-4A and -4B) fusion (F) proteins were cloned and sequenced. The predicted amino acid sequences of the F proteins had similar characteristic traits to those reported for the F proteins of other paramyxoviruses. They were more closely related to the F proteins of simian virus 5 (SV5), mumps virus (MuV), hPIV-2 and Newcastle disease virus (NDV) than to the F proteins of hPIV-1, hPIV-3, Sendai virus (SV) and measles virus (MV). In addition, hPIV-4A, hPIV-4B, SV5 and MuV shared a common feature of genomic organization: there was a small ORF between the F and haemagglutinin-neuraminidase (HN)-coding sequences, implying a common ancestry.

Structure-function analysis of the Sendai virus F and HN cytoplasmic domain: different role for the two proteins in the production of virus particle.

The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glycoproteins in the process of virus assembly and budding are evaluated. Recombinant Sendai virus (rSeV) mutants are generated carrying modifications in the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state (35)[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cell supernatants are measured relative to the extent of the infection, assessed by the intracellular N protein signal. For both the F and HN cytd truncation mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutants, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this threshold is reached up appears to depend on the nature of the F cytd. A minimal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd truncations result in significant reduction of virion production. On the other hand, a normal virion production can take place with an underrepresentation of or, even, an undetectable HN in the particles. The HN uptake in virion is confirmed to depend on the previously proposed cytd SYWST signal (T. Takimoto, T. Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Virol. 72, 9747-9754.).

HN proteins of human parainfluenza type 4A virus expressed in cell lines transfected with a cloned cDNA have an ability to induce interferon in mouse spleen cells.

Primary monkey kidney cells infected with human parainfluenza type 4A virus (HPIV-4A) were treated with various concentrations of formaldehyde. Formaldehyde (0.275%) treatment completely blocked virus production. However, when mouse spleen cells were cocultured with the fixed virus-infected cells, interferon was produced in the culture fluid. On the other hand, when mouse spleen cells were incubated with the fixed virus-infected cells in the presence of anti-HPIV-4A antiserum or a mixture of anti-HN protein monoclonal antibodies, interferon activity could scarcely be detected in the culture fluid. These findings indicated that the fixed virus-infected cells had an ability to induce interferon in mouse spleen cells and that the HN protein was related to interferon induction. Subsequently, a recombinant plasmid was constructed by inserting the cDNA of the HN gene of HPIV-4A into a pcDL-SR alpha expression vector. Mouse spleen cells produced interferon when cocultured with COS7 cells transfected with the recombinant plasmid, but did not when cocultured with COS7 cells transfected with the vector alone. Furthermore, we established HeLa cells constitutively expressing HPIV-4A HN (HeLa-4aHN cells) or F protein (HeLa-4aF cells). Type I (alpha/beta) interferon was detected in culture fluids of mouse spleen cells with HeLa-4aHN cells, but was not detected in those with HeLa-4aF cells. Therefore, it was concluded that the HN glycoproteins on the cell surface were sufficient for interferon induction to occur.

Sendai virus-based expression of HIV-1 gp120: reinforcement by the V(-) version.

BACKGROUND: We have established a system for recovering Sendai virus (SeV), a nonsegmented negative strand RNA virus, entirely from cDNA at an extremely high rate, and have succeeded in creating a V(-) SeV whose gene expression was greatly enhanced by the deletion of the nonessential V gene. Because of its extreme medical importance, there has been a strong need for the establishment of a better system to express the gp120 envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) in sufficient quantity and purity. It also remains to be established to produce gp120 in in vitro natural host cells for HIV-1 such as human primary blood mononuclear cells, macrophages or established T cell lines. RESULTS: Using the above system, we created recombinant Sendai viruses expressing the gp120 in CV1 cells, a monkey kidney line. The expression level from the standard V(+) version has already reached 2.2/microg per 10(6) infected cells, which was readily purified from the culture fluid with a recovery rate of about 60%, and has so far appeared to be functionally and serologically authentic. The inserted gp120 gene was stably maintained during numerous passages of the recombinant virus. The V(-) version-based expression was even more robust, consistently reaching over 6.0 microg per 10(6) cells, a level that is one of the highest currently attainable for gp120 production in mammalian cells. Furthermore, a broad host range of SeV allowed gp120 production in all the three natural host cells for HIV-1 described above. CONCLUSIONS: SeV-based expression serves as a novel choice for producing large quantities of HIV-1 gp120 and will greatly facilitate biochemical, biological and immunological studies of this important glycoprotein.

The attachment protein of Hendra virus has high structural similarity but limited primary sequence homology compared with viruses in the genus Paramyxovirus.

The complete nucleotide sequence of the attachment protein gene of Hendra virus, a new member of the subfamily Paramyxovirinae, has been determined from cDNA clones derived from viral genomic RNA. The deduced mRNA is 2565 nucleotides long with one open reading frame encoding a protein of 604 amino acids, which is similar in size to the attachment protein of the members of the subfamily. However, the mRNA transcript is >600 nucleotides longer than others in the subfamily due to the presence of long untranslated regions at both the 5' and 3' ends. The protein is designated G because it lacks both hemagglutination and neuraminidase activities. It contains a hydrophobic transmembrane domain close to the N terminus, eight potential N-linked glycosylation sites, and 18 cysteine residues. Although the HeV G protein had low sequence homology with Paramyxovirinae members, the predicted folding pattern of its extracellular globular head was very similar to that of members of the genus Paramyxovirus, with the location of seven potential pairs of sulfide bonds absolutely conserved. On the other hand, among the seven residues known to be critical for neuraminidase activity, only one was conserved in the Hendra virus G protein compared with at least six in HN proteins of paramyxoviruses and rubulaviruses and four in H proteins of morbilliviruses. The biological significance of this finding is discussed.