Sendai Virus Fusion Protein Research Articles

Transfection of Sendai virus F gene cDNA with mutations at its cleavage site and HN gene cDNA into COS cells induces cell fusion.

In contrast to the wild type Sendai virus fusion protein (F), a mutated F to possess a cleavage site similar to that of virulent Newcastle disease virus F, could be cleaved by proteases present in COS cells. When mutated F and hemagglutinin-neuraminidase (HN) were coexpressed at the cell surface, syncytium formation was observed.

Effect of substitution of hemagglutinin-neuraminidase with influenza hemagglutinin on Sendai virus F protein mediated membrane fusion

Recombinant virosomes containing fusion protein (F) of Sendai virus and the envelope glycoproteins hemagglutinin (HA) and neuraminidase (NA) of influenza virus within the same membrane were prepared. Such hybrid vesicles were found to hemolyse red blood cells both at pH 7.4 and pH 5.0. Hemolysis induced by hybrid vesicles was much higher than seen with F-virosomes in the presence of WGA, but was about two-fold less than the hemolysis caused by F,HN-virosomes. Reconstituted influenza virus envelopes and F-virosomes failed to induce hemolysis at pH 7.4. Using a fluorescence probe-based lipid mixing fusion assay, hybrid virosomes were found to fuse with cultured HeLa cells both at pH 7.4 as well as pH 5.0. The data indicate that the presence of Sendai virus HN protein in the virosomal membrane is not absolutely essential for the virosome-cell fusion process.

Mice Lacking CD8 + T Cells Develop Greater Numbers of IgA-Producing Cells in Response to a Respiratory Virus Infection

Antibody production profiles have been compared for Sendai virus infection of normal mice and mice that lack CD8 + T cells as a consequence of treatment with a lymphocyte subset-specific monoclonal antibody or homozygous disruption of the β 2-microglobulin (β2-m(-/-)) gene encoding the light chain of the class I major histocompatibility complex glycoprotein. Using the single-cell ELISPOT assay, we show a relative increase in IgA antibody forming cell (AFC) numbers in the mediastinal lymph node (MLN), spleen, and bone marrow of the CD8-depleted mice. This is reflected in higher serum IgA titers. Similarly, secondary infection with a large dose of Sendai virus leads to greater prevalence of virus specific IgA AFCs as early as Day 5 postinfection in the β2-m(-/-) mice. Also, in primed β2-m(-/-) mice challenged with vaccinia constructs containing the genes for the hemagglutinin-neuraminidase (HN), nuclear protein, or the fusion protein of Sendai virus, the majority of the virus-specific AFCs in the MLN are specific for HN and secrete IgA.

Expression of the Sendai Virus Fusion Protein in Insect Cells and Characterization of its Post-translational Modifications

The fusion (F) protein of Sendai virus was expressed with a baculovirus system in insect cells. The F protein is synthesized in its uncleaved form F0, which appears in SDS-containing gels as two bands with M(r) values of 66K and 64K. Digestion of the F protein with endoglycosidase H revealed that the 66K species contains high mannose-type carbohydrates, whereas partially processed oligosaccharides are attached to the 64K species. Pulse-chase experiments showed that the F protein is initially synthesized as its 66K form. After 1 h of chase this precursor glycoprotein is partially converted to the 64K species and exposed at the cell surface. Other modifications of the F protein in insect cells are the formation of intramolecular disulphide linkages and oligomerization. However, processing and plasma membrane transport of the F protein in insect cells are incomplete and retarded compared to the F protein synthesized in Sendai virus-infected mammalian cells.

Interaction of fluorescently labeled analogues of the amino-terminal fusion peptide of Sendai virus with phospholipid membranes.

A peptide representing the NH2-terminal (33 amino acid residues) of the fusion protein (F) of Sendai virus, as well as its Gly12-->Ala12 mutant, were synthesized, fluorescently labeled, and spectroscopically and functionally characterized. Peptide-induced vesicle fusion was demonstrated by a combination of increased visible absorbance, lipid mixing assay, and electron microscopy. Both peptides, with the mutant peptide being significantly more potent, were shown to induce membrane fusion and bilayer perturbation of negatively charged phospholipid vesicles. These results are consistent with a previous study that showed that a similar mutation in the homologous NH2-terminal segment of simian virus 5 greatly enhanced syncytium formation (Horvath, C. M., and Lamb, R. A. (1992) J. Virol. 66, 2443-2455). Circular dichroism spectroscopy revealed similar high alpha-helical contents of both peptides in methanol and in trifluoroethanol. Using fluorescently labeled peptide analogues we found that (i) the peptides' membrane partition coefficients are in the range of 10(5) M-1; (ii) the NH2 terminus of the wild-type peptide is located within the lipid bilayer, whereas that of the variant peptide lies on the surface; and (iii) both peptides tend to self-associate in their membrane-bound state. The results support a model in which an alpha-helical secondary structure and self-aggregation of peptides are necessary conditions for membrane fusion. The observed differences in the peptides' fusogenic abilities are hypothesized to result from differences in the peptides' degree of penetration into the membrane, induction of membrane destabilization, and ability to cause vesicles to aggregate. The data support Sendai virus-cell fusion models in which the fusion peptide plays a crucial role in fusion induction by destabilizing the bilayer and by triggering the association of viral fusion protein molecules.

Assessment of Membrane Fusion Efficiency and Its Use for Distinguishing Epitopes on the Fusion (F) Protein of Sendai Virus (HVJ)

Upon incubation of radioiodinated Sendai virus with Ehrlich ascites tumor cells to induce viral envelope-cell membrane fusion, TCA-soluble radioactivity was found to be rapidly released from the virus into the medium. The amount of the TCA-soluble 125I species released into the medium was correlated with the extent of envelope-membrane fusion. Using a new method based on this phenomenon, we evaluated the fusion-inhibiting activity of monoclonal anti-F protein antibodies and could distinguish epitopes within partially overlapping regions of the F protein by their effects on fusion activity. We concluded this method is a reliable and sensitive one for measuring envelope-membrane fusion.

Changes in specific cleavability of the Sendai virus fusion protein: implications for pathogenicity in mice.

Sendai virus mutants, KDe-21 and KDe-62, which had undergone multiple cycles of replication in Madin Darby canine kidney (MDCK) cells in the absence of exogenous proteases were isolated. The fusion (F) protein of the mutants regained proteolytic cleavability in MDCK cells and chick embryos, but the F protein remained non-cleavable in other cell lines. Unlike the F protein of wild-type (wt) virus, the mutant F was resistant to trypsin but was sensitive to elastase and, to a lesser extent, to chymotrypsin. Sequence analyses of the F gene and the F protein revealed an amino acid substitution at the cleavage site, Arg(116) to Ile, which conferred trypsin resistance and enhanced cleavability at Ile(116) by elastase and host proteases present in MDCK cells and in chicken embryos. In contrast to the pneumopathogenicity in mice of wt Sendai virus, the KDe mutants were non-pathogenic; cleavage activation of the F protein did not occur in the lungs and thereby infection was terminated after an initial cycle of replication.

Construction of expression plasmids for the fusion protein of Sendai virus, and their expression in E. coli cells and eucaryotic cells

To examine the properties and the role of the fusion protein (F) of Sendai virus at the molecular level, a plasmid, pUC-F, was constructed by inserting cDNA for the F protein into a pUC vector. Upon induction of E. coli cells transformed with pUC-F, a new protein was obtained, which was identified as Fo on Western blot analysis. The cDNA fragment for the F gene was excised from pUC-F and inserted into an eucaryotic expression vector, pSVL, to yield pSVL-F. COS-1 cells transfected with pSVL-F gave a band on SDS-gel electrophoresis which corresponded to the size of the Fo protein.

Conformation and stability of Sendai virus fusion protein

The conformation and stability of Sendai virus fusion (F) protein were studied by circular dichroism spectroscopy, and the protein predictive models of Chou and Fasman and Robson and Suzuki were used to elucidate the secondary structure of Sendai virus F protein. The F protein conformation is predicted to contain 33% α-helix, 53% β-sheet and 15% β-turn by the Chou and Fasman model, and 30% α-helix, 55% β-sheet, 9% β-turn and 7% random coil by the Robson and Suzuki model, C.d. studies of F protein purified in the presence of the non-ionic detergent, n-octylglucoside, indicated the presence of 49% α-helix and 31% β-sheet at pH 7.0, 54% α-helix and 28% β-sheet at pH 9.0 and 50% α-helix and 23% β-sheet at pH 5.4. A small change in conformation of the protein occurred when the pH was titrated from 7.0 to 5.4 and from 7.0 to 9.0 and a more pronounced conformational change occured when the pH was changed from 9.0 to 5.4. The F protein in 0.2% n-octylglycoside was resistant to denaturation by 4 m guanidine hydrochloride, the reducing agent 20 m m mercaptoethanol, and to increases in temperature from 5 to 80°C. Monoclonal anti-F protein antibody showed an increased binding to whole virus when the pH was changed from 7.0 to 9.0. The antibody binding was decreased when the pH was shifted from 9.0 to 5.4. Maximum haemolytic activity was observed with virus that was preincubated at pH 8.0. The enhanced haemolytic activity could be correlated with a small conformational change in the fusion protein at basic pH, as determined by the c.d. spectrum and the antibody binding data from enzyme-linked immunoadsorbent assay. These results represent the first report of a conformational change in Sendai virus F protein when the pH was changed from basic to acidic pH.

Comparison of non-ionic detergents for extraction and ion-exchange high-performance liquid chromatography of sendai virus integral membrane proteins

The integral membrane proteins of Sendai virus haemagglutinin-neuraminidase (HN) and fusion protein (F) were extracted from purified virions with 2% of a non-ionic detergent, i.e., polyoxyethylene alkyl ethers varying by 8–14 hydrocarbon units in the alkyl chain and by 4–8 ethylene glycol units in the oxyethylene chain. Triton X-100 and octyl glucoside were included as reference detergents. The hydrophile-lipophile balance (HLB) and the critical micelle concentration (CMC) of the detergents were determined. A decrease in length of the oxyethylate by 8-5 ethylene glycol units and an increase in the alkylate by 8–12 hydrocarbon units resulted in higher yields of extracted proteins. The highest yields were obtained for C 12E 5 with an HLB of 11.7. Yields of extracted protein could be correlated with the HLB values of the polyoxyethylene alkyl ethers. The structural integrity of HN and F was not affected during extraction by either detergent as measured by their reactivity with monoclonal antibodies directed against native HN and F. Extracts were subjected to anion-exchange high-performance liquid chromatography (HPLC) on a Mono Q column in the presence of 0.1% of the detergent used for extraction. Eluate fractions were analysed by sodium dodecyl polyacrylamide gel electrophoresis and recoveries of HN and F protein were determined by size-exclusion HPLC. The immunological activity of HN and F was tested in an enzyme-linked immunosorbent assay. The highest recoveries of HN and F (80%) were obtained with C 10E 5 in the elution buffer. HN and F were partially purified and the immunllogical activity was well preseved.

Protease activation mutants of sendai virus: Sequence analysis of the mRNA of the fusion protein (F) gene and direct identification of the cleavage-activation site

Trypsin cleaves the fusion protein (F) of wild-type Sendai virus into two disulfide-linked polypeptides, F 1 and F 2, and thereby activates the membrane fusion activity of the virus. A. Scheid and P. W. Choppin ((1976). Virology, 265–277) selected mutant viruses of which the F protein could be activated by different proteases, either elastase, chymotrypsin, or plasmin. Herein, we have further characterized five of these mutants. Sequencing of each mutant mRNA encoding the 60–70 amino acids surrounding the cleavage site revealed one or two amino acid changes near or at the cleavage sites. Virions cleaved in vitro by the appropriate proteases were assayed of their fusion activity by hemolysis, and the cleavage sites were determined by amino acid sequencing. In three cases, the change of protease specificity can be accounted for by changed amino acids right at the cleavage site, whereas several other mutations that potentiate cleavage at new sites by new proteases are somewhat removed from the actual cleavage site. We surmise that such mutations might alter local polypeptide conformation, thereby allowing the proteases access to existing sites. Cleavage at new sites produced fusion proteins with novel F 1 NH-termini. We found that a mutant with a charged residue at the third position of this normally hydrophobic NH-terminal sequence retains activity in the hemolysis assay, whereas a mutant with a charged residue at the first position does not.

Aspects of the molecular interactions between the fusion protein of sendai virus and its target membrane

Aspects of the molecular interactions between the fusion protein of sendai virus and its target membrane

Activation of the Sendai virus fusion protein (f) involves a conformational change with exposure of a new hydrophobic region.

The F protein of paramyxoviruses is actively involved in the induction of membrane fusion. This fusion may be between viral and cellular membranes, as in the initiation of infection or in virus-induced lysis of erythrocytes, or between the plasma membranes of different cells. The F protein is activated by proteolytic cleavage to yield two disulfide-linked polypeptides (F1 and F2); however, its mechanism of action is not clear. In the present study, the conformations of the inactive, uncleaved precursor of glycoprotein (F0), and the active, cleaved form (F1,2) have been compared. The UV circular dichroism spectra of the two forms of the F protein indicate that cleavage results in a conformational change. Detergent-binding studies by velocity sedimentation analysis of Triton X-100-protein complexes revealed an increase in exposed hydrophobic surface of the protein on cleavage. The inactive F0 bound an estimated 27 molecules of Triton X-100/F polypeptide; these molecules are presumably bound to the hydrophobic region of the glycoprotein that anchors the spike-like protein in the virus membrane and that is common to both forms of F. The active form, F1,2, bound 67 molecules of Triton X-100. This increase in the number of detergent binding sites upon F protein activation indicates the presence of a hydrophobic region that is peculiar to the active form, and that may be of functional significance in the membrane fusion reaction.

Purification of the fusion protein of Sendai virus: analysis of the NH2-terminal sequence generated during precursor activation.

The two glycoproteins of Sendai virus, the hemagglutinin-neuraminidase and the fusion protein (F), were separated and purified by affinity chromatography on a Lens culinaris lectin-Sepharose column. F was shown to consist of two disulfide-bonded glycopolypeptide chains, F1 and F2, of molecular weights 51,000 and 11,000, each of which contained 15% carbohydrate by weight. Amino-terminal sequence analysis showed that F2 was blocked and that the hydrophobic sequence NH2-Phe-Phe-Gly-Ala-Val-Ile-Gly-Ile-Ile-Ala-Leu-Gly-Pro-Ala-Thr- was at the amino terminus of F1. This sequence shows identity at six positions with the hydrophobic amino-terminal sequence of the smaller glycopolypeptide chain, HA2, of the hemagglutinin of influenza virus. Both F1 and HA2 are formed by proteolytic cleavage of precursor glycoproteins (Fo, Sendai virus; HAo, influenza virus). Since these cleavages confer infectivity upon both Sendai and influenza viruses and the ability to induce cell-to-cell fusion upon Sendai virus, the hydrophobic NH2-terminal sequences on F1 and HA2 may play a role in fusion of viral and host-cell membranes.