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.

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.

Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs.

Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

Structural features of paramyxovirus F protein required for fusion initiation.

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

Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer.

We previously demonstrated that gene replacement of mouse macrophage metalloelastase (MME) into murine melanoma cells that grow rapidly and are MME deficient suppresses the primary tumor growth in vivo by halting angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer using a cDNA-encoding MME gene. In a subcutaneous tumor model of CT-26 mouse colon cancer cells that are MME deficient, syngeneic mice repetitively treated with direct injections into the tumors of MME- hemagglutinating virus of Japan (HVJ), a type of HVJ-cationic liposome encapsulating a plasmid expressing MME, developed smaller tumors (210 +/- 47.2 mm(3) versus 925 +/- 156 mm(3) mean +/- SEM; p = 0.0004) with fewer microvessels (10.25 +/- 1.03 vs. 17.25 +/- 2.14; p = 0.03) than control mice. TUNEL staining revealed a significant increase of apoptotic cells in the MME-HVJ liposomes-treated tumors compared with control tumors. MME was effectively expressed in the s.c. tumors treated with MME-HVJ liposomes, inducing angiostatin generation in those tumors, as demonstrated by Western blot analysis. In conclusion, our study demonstrated that repeated in vivo transduction of the MME gene directly into the tumors using HVJ-cationic liposomes suppressed the tumor growth by an antiangiogenic mechanism, providing, then, a feasible strategy for gene therapy of cancer.

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.

The Sendai virus membrane fusion mechanism studied using image correlation spectroscopy.

The mechanism of Sendai virus membrane fusion to cultured cell membranes was studied. Viral lipids were labeled with the lipophilic dye, 4-(4-(dihexadecylamino)styryl-N-methylquinolinium iodine) (DiQ), and viral proteins were labeled using fluorescein isothiocyanate (FITC). The redistribution of these probes from the virus to cultured cells was followed using the technique of image correlation spectroscopy. This technique assayed the intensity change and the redistribution of these probes as fusion progressed from a more to less aggregated state. The lipid probe DiQ dispersed into the membrane of the target membrane at both 22 and 37 degrees C, while the FITC-labeled proteins dispersed only at 37 degrees C. Simultaneous labeling of virus with both of these probes showed that at 37 degrees C their redistribution proceeded at different rates. These data were consistent with the formation of a hemifusion intermediate during the fusion process.

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.

Sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition.

Viral glycoproteins catalyze the fusion between viral and cellular membranes. The fusion protein (F(1)) of Sendai virus has two fusion peptides. One is located at its N-terminus and the other, highly homologous to the HIV-1 and RSV fusion peptides, in the interior of the F(1) protein. A synthetic peptide corresponding to the internal fusogenic domain, namely, SV-201, was found to inhibit virus-cell fusion without interfering with the binding of the virus to the target cells, thus highlighting the importance of this region in Sendai virus-induced membrane fusion. However, its detailed mechanism of inhibition remains unknown. Here, we synthesized a shorter version of SV-201, namely, SV-208, an elongated one, SV-197, and two mutants of SV-201, and compared them functionally and structurally with SV-201. In contrast to SV-201, SV-208 and the two mutants do not inhibit virus-cell fusion. The differences in the oligomerization state of these peptides in aqueous solution and within the membrane, and in their ability to bind to Sendai virions, enabled us to postulate a possible mechanism of viral entry inhibition: SV-201 binds to its target in Sendai virions before the F(1) internal fusion peptide binds to the membrane, therefore blocking the F(1) conformational change required for fusion. In addition, we further characterized the fusogenic activity of the internal fusion peptide, compared to the N-terminal one, and determined its structure in the membrane-bound state by means of fluorescence, CD, and ATR-FTIR spectroscopy. Remarkably, we found that SV-201 and its elongated form, SV-197, are highly potent in inducing fusion of the highly stable large unilamellar vesicles composed of egg phosphatidylcholine, a property found only in an extended version of the HIV-1 fusion peptide. The inhibitory activity of SV-201 and its fusogenic ability are discussed in terms of the "umbrella" model of Sendai virus-induced membrane fusion.

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.

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.

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.).

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.

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.

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

Identification of nucleocapsid protein residues required for Sendai virus nucleocapsid formation and genome replication.

Alanine substitution mutations in the Sendai virus nucleocapsid (NP) protein have defined highly conserved hydrophobic and charged residues from amino acids (aa) 362 to 371 that are essential for function of the protein in RNA replication. Mutant NP362, which had the change F362A, was incapable of supporting in vitro RNA replication. NP362 expressed alone formed extended oligomers which exhibited an abnormal morphology and density suggesting that these particles were not associated with any RNA. Mutant NP364, which had changes L362A and G365A, was also inactive in RNA replication; however, this was because the protein was unstable and did not form NP-NP complexes. Mutant NP370 mutant, which had changes K370A and D371A, was inactive in in vitro replication, although it could form the required NP0-P and NP-NP protein complexes. The self-assembled nucleocapsid-like particles formed by NP370 alone had a morphology like that of wild-type NP and banded in CsCl as ribonucleoprotein particles, suggesting that they contained cellular RNA. These data suggest that the replication defect of NP370 may be in the ability to specifically encapsidate Sendai virus genome RNA. Mutant NP373, where nonconserved charged residues at aa 373 and 375 were substituted with alanine, gave a wild-type phenotype. Thus these amino acids are not required for either protein-protein interactions or in vitro Sendai virus RNA replication.

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.

Structural basis for paramyxovirus-mediated membrane fusion.

Paramyxoviruses are responsible for significant human mortality and disease worldwide, but the molecular mechanisms underlying their entry into host cells remain poorly understood. We have solved the crystal structure of a fragment of the simian parainfluenza virus 5 fusion protein (SV5 F), revealing a 96 A long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Six amino acids, potentially part of the fusion peptide, form a segment of the central coiled coil, suggesting that this structure extends into the membrane. Deletion mutants of SV5 F indicate that putative flexible tethers between the coiled coil and the viral membrane are dispensable for fusion. The lack of flexible tethers may couple a final conformational change in the F protein directly to the fusion of two bilayers.

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.

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.