Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.     

Sendai virus F glycoprotein induces IL-6 production in dendritic cells in a fusion-independent manner

We previously reported that inactivated Sendai virus particle (hemagglutinating virus of Japan envelope; HVJ-E) has anti-tumor effects by eliciting IL-6 production in dendritic cells (DCs). In the present study, we investigated which components of HVJ-E elicit IL-6 production. HVJ-E containing F0 protein inactive for virus envelope-cell membrane fusion enhanced IL-6 production. Reconstituted liposomes containing F protein stimulated IL-6 production. The antibody against F protein inhibited IL-6 secretion by HVJ-E. When carbohydrate chains of the F glycoprotein were removed, HVJ-E lost the ability to stimulate IL-6 secretion. These results suggest that F glycoprotein is required for IL-6 production in DCs.     

Sendai virus M protein binds independently to either the F or the HN glycoprotein in vivo.

We have analyzed the mechanism by which M protein interacts with components of the viral envelope during Sendai virus assembly. Using recombinant vaccinia viruses to selectively express combinations of Sendai virus F, HN, and M proteins, we have successfully reconstituted M protein-glycoprotein interaction in vivo and determined the molecular interactions which are necessary and sufficient to promote M protein-membrane binding. Our results showed that M protein accumulates on cellular membranes via a direct interaction with both F and HN proteins. Specifically, our data demonstrated that a small fraction (8 to 16%) of M protein becomes membrane associated in the absence of Sendai virus glycoproteins, while > 75% becomes membrane bound in the presence of both F and HN proteins. Selective expression of M protein together with either F or HN protein showed that each viral glycoprotein is individually sufficient to promote efficient (56 to 73%) M protein-membrane binding. Finally, we observed that M protein associates with cellular membranes in a time-dependent manner, implying a need for either maturation or transport before binding to glycoproteins.     

Characterization of the fusogenic properties of Sendai virus: kinetics of fusion with erythrocyte membranes

A novel fluorescence assay [Hoekstra, D., De Boer, T., Klappe, K., & Wilschut, J. (1984) Biochemistry 23, 5675-5681] has been used to characterize the fusogenic properties of Sendai virus, using erythrocyte ghosts and liposomes as target membranes. This assay involves the incorporation of the "fusion-reporting" probe in the viral membrane, allowing continuous monitoring of the fusion process in a very sensitive manner. Fusion was inhibited upon pretreatment of Sendai virus with trypsin. Low concentrations of the reducing agent dithiothreitol (1 mM) almost completely abolished viral fusion activity, whereas virus binding was reduced by ca. 50%, indicating that the fusogenic properties of Sendai virus are strongly dependent on the integrity of intramolecular disulfide bonds in the fusion (F) protein. Pretreatment of erythrocyte ghosts with nonlabeled Sendai virus inhibited subsequent fusion of fluorophore-labeled virus irrespective of the removal of nonbound virus, thus suggesting that the initial binding of the virus to the target membrane is largely irreversible. As a function of pH, Sendai virus displayed optimal fusion activity around pH 7.5-8.0. Preincubation of the virus at suboptimal pH values resulted in an irreversible diminishment of its fusion capacity. Since virus binding was not affected by the pH, the results are consistent with a pH-induced irreversible conformational change in the molecular structure of the F protein, occurring under mild acidic and alkaline conditions. In contrast to virus binding, fusion appeared to be strongly dependent on temperature, increasing ca. 25-fold when the temperature was raised from 23 to 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell-cell fusion and a decreased dependency on the HN attachment protein for activity

Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.     

Isolation of 3-(4,8,12-Trimethyltridecyl)-furan (“Phytofuran”) from Burley Tobacco

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

Construction and characterization of a fluorescent sendai virus carrying the gene for envelope fusion protein fused with enhanced green fluorescent protein

Sendai virus (SeV) is an enveloped virus with a non-segmented negative-strand RNA genome. SeV envelope fusion (F) glycoproteins play crucial roles in the viral life cycle in processes such as viral binding, assembly, and budding. In this study, we developed a viable recombinant SeV designated F-EGFP SeV/ΔF, in which the F protein was replaced by an F protein fused to EGFP at the carboxyl terminus. Living infected cells of the recombinant virus were directly visualized by green fluorescence. The addition of EGFP to the F protein maintained the activities of the F protein in terms of intracellular transport to the plasma membrane via the ER and the Golgi apparatus and fusion activity in the infected cells. These results suggest that this fluorescent SeV is a useful tool for studying the viral binding, assembly, and budding mechanisms of F proteins and the SeV life cycle in living infected cells.     

A 45,000-M(r) glycoprotein in the Sendai virus envelope triggers virus-cell fusion.

Sendai virus envelopes devoid of hemagglutinin-neuraminidase but containing the fusion protein (F-virosomes) were prepared. F-virosomes exhibited discernible serine protease activity at neutral pH. Electrophoretic analysis of the protein profile of the F-virosomes under nonreducing conditions, by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, led to the identification of a previously unknown glycoprotein with a relative molecular weight of 45,000 (45K protein) associated with the F protein. The identity of the 45K protein, as distinct from F protein, was established by Western blot analysis with F- and 45K-specific antibodies. This 45K protein forms a nexus with the F protein through noncovalent hydrophobic interactions, as proved by its sensitivity to urea treatment, and it is essential for the proteolytic activity of the F-virosomes as well as for the fusion of the viral envelope with host cell membrane. N-terminal sequence analysis (first 11 amino acids) of this protein showed strong homology (> 90%) to flavivirus NS3 serine proteases but no similarity to any of the Sendai viral proteins. On the basis of the N-terminal sequence, oligonucleotides were designed corresponding to the sense and antisense DNA sequences. Dot blot hybridization and primer extension with these oligonucleotides with the viral and the host genome confirmed the host origin of this protein. Further, the limited proteolytic digestion of the target membrane resulted in significant inhibition of viral fusion with it. On the basis of these results, we postulate a model for the molecular mechanism of F protein-induced membrane fusion, which may provide a rationale for other paramyxoviruses.     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.     

The activity of membranes reconstituted from HVJ envelope proteins and lipids to induce hemolysis and fusion between liposomes and erythrocytes

A simple method for preparation of lipid-free envelope proteins (HN protein and F protein) of HVJ (Sendai virus) was developed. Reconstituted 'envelopes' were then prepared from envelope proteins and various lipids by the detergent dialysis method, and the activity to induce hemolysis and fusion between liposome and erythrocyte was studied. Lipid-free envelope protein aggregates could induce hemolysis and liposome-erythrocyte fusion. The activity was however greatly augmented by incorporation of envelope proteins into membrane of viral total lipids. Hemolytic and fusogenic activity was somewhat augmented by incorporation of envelope proteins into dipalmitoylphosphatidylcholine/cholesterol (1:1, molar ratio) and dimyristoylphosphatidylcholine/cholesterol (1:1), though the augmentation was lower than that observed with viral total lipids. When 'envelopes' were reconstituted with the proteins and viral total lipids supplemented with phosphatidylethanolamine, two kinds of 'envelopes' were prepared; one was permeable to Dextran (Mr 75000) and hemolytic, and the other was impermeable to Dextran and nonhemolytic. The latter acquired hemolytic activity after subjection to freezing and thawing, and its barrier function was lost concomitantly. The study suggests that envelope proteins (HN protein and F protein) could function without lipids but their activity was greatly influenced by not only the composition of additional lipids but also mode of arrangement of components on the reconstituted membranes.     

Polarized distribution of viral envelope proteins in the plasma membrane of infected epithelial cells

The surface distribution of the envelope glycoproteins of influenza, Sendai and Vesicular Stomatitis viruses was studied by immunofluorescence and immunoelectromicroscopy in infected epithelial cell monolayers, from which these viruses bud in a polarized fashion. It was found that before the onset of viral budding, the envelope proteins are exclusively localized into the same plasma membrane domains of the epithelial cells from which the virions ultimately bud: the glycoproteins of influenza and Sendai were detected at the apical surface, while the G protein of Vesicular Stomatitis virus was concentrated at the basolateral region. On the other hand, Sendai virus nucleocapsids, which can be easily identified in the cytoplasm before viral assembly, could be observed throughout the cell, not showing any preferential localization near the surface that the virions utilize for budding. These results are consistent with a model in which the asymmetric distribution of viral envelope proteins, rather than a polarized delivery of nucleocapsids, directs the polarity of viral budding. Furthermore, the asymmetric surface localization of viral glycoproteins suggests that these proteins share with intrinsic surface proteins of epithelial cells common biogenetic mechanisms and informational features or "sorting out" signals that determine their compartmentalization in the plasma membrane.     

Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells

A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells.     

Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion

Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.     

Identification of ectopic anionic trypsin I in rat lungs potentiating pneumotropic virus infectivity and increased enzyme level after virus infection.

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. In search of such target processing proteases in the airway, we recently found a new candidate trypsin-like processing protease in rat lungs, which was induced by Sendai virus infection, and identified as ectopic rat anionic trypsin I. On SDS/PAGE under reducing and nonreducing conditions, the purified enzyme gave protein bands corresponding to 29 and 22 kDa, respectively, i.e. at the same positions as rat pancreatic anionic trypsin I. It exhibited an apparent molecular mass of 31 kDa on molecular sieve chromatography and its isoelectric point was pH 4.7. The amino-acid sequences of the N-terminus and proteolytic digest peptides of the purified enzyme were consistent with those of rat pancreatic anionic trypsin I. Its substrate specificities and inhibitor sensitivities were the same as those of the pancreatic enzyme. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and hemagglutinin of human influenza A virus, and potentiated the infectivity of Sendai virus in the same dose-dependent manner as the pancreatic one. Immunohistochemical studies revealed that this protease is located in the stromal cells in peri-bronchiolar regions. These results suggest that ectopic anionic trypsin I in rat lungs induced by virus infection may trigger virus spread in rat lungs.     

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

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

A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.     

Trypsin action on the growth of Sendai virus in tissue culture cells. IV. Evidence for activation of sendai virus by cleavage of a glycoprotein.

Results obtained by using a reconstitution technique on the Sendai virus envelope confirm that cleavage of one of the envelope glycoproteins (GP2) is prerequisite for activation of hemolytic and cell fusion activities of Sendai virus. The cleavage of GP2 occurs even when free envelope subunits are directly treated with trypsin in the presence of detergent. Trypsin treatment, either of the reconstituted particle or of the free envelope subunits but not of the intact virion, also causes a cleavage of the largest envelope glycoprotein (GP1), suggesting that a site on GP1 sensitive to trypsin becomes exposed during solubilization and reconstitution. The latter cleavage, however, is not associated with any changes in biological activities.     

Pneumopathogenicity of a Sendai virus protease-activation mutant, TCs, which is sensitive to trypsin and chymotrypsin.

A protease-activation mutant of Sendai virus, TCs, was isolated from a trypsin-resistant mutant, TR-5. TCs was activated in vitro by both trypsin and chymotrypsin. TCs was, however, less sensitive to trypsin and chymotrypsin than were the wild-type virus and TR-5, respectively. F protein of TCs had a single amino acid substitution at residue 114 from glutamine to arginine, resulting in the appearance of the new cleavage site for trypsin and the shift of the cleavage site for chymotrypsin. Activation of TCs in the lungs of mice occurred less efficiently than that of the wild type, and TCs caused a less severe pneumopathogenicity than did the wild-type virus, which supports our previous view that the in vitro trypsin sensitivity of Sendai virus can be a good indication of pneumopathogenicity in mice.     

Interactions Between Sendai Virus and Human Erythrocytes

Concentrated Sendai virus, when adsorbed to erythrocytes at 4 C, caused invaginations in the plasma membrane. Following elevation of the temperature to 37 C, the plasma membrane became fused with the viral envelope before dissolution of the virions and rupture of the cells. Cell lysis was accompanied by rapid and total loss of hemoglobin to the extracellular space. Following aqueous pyridine extraction, the hemoglobin-free ghosts remaining were found to be devoid of N-acetylneuraminic acid and to have solubility properties different from those of normal erythrocyte ghosts. By the action of viral neuraminidase, bound N-acetylneuraminic acid was also liberated from purified virus receptor substance whose electrophoretic mobility was thereby substantially reduced. Cu⁺⁺ selectively inhibited hemolysis and neuraminidase without interfering with hemagglutination and attachment. Neuraminidase appeared to be essential for Sendai virus hemolysis; viral particle size may also be a critical factor in this process.     

Accumulation of Sendai virus glycoproteins in cell-cell contact regions and its role in cell fusion.

Lateral motion of the viral envelope proteins in the target cell membrane was shown recently to be essential for cell fusion by Sendai virus (Henis, Y. I., Herman-Barhom, Y., Aroeti, B., and Gutman, O. (1989) J. Biol. Chem. 264, 17119-17125). To explore the mechanism that gives rise to this requirement, we have now investigated the distribution of Sendai virus envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) on human erythrocytes in the course of fusion, using fluorescence microscopy and image analysis. In these studies, both the F and the HN proteins were found to accumulate in cell-cell contact regions, on the time scale of the fusion process. We propose that migration of the viral glycoproteins to cell contact regions and accumulation at the contact sites are essential parts of the fusion mechanism and form the basis to the requirement for their lateral motion in the fusion event.