Andreas Scheid

Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus

Abstract The glycoproteins of Sendai virus have been isolated by a procedure involving extraction with the nonionic detergent Triton X-100 and affinity chromatography on fetuin-Sepharose. The largest Sendai virus glycoprotein (MW ~69,000) possesses both hemagglutinating and neuraminidase activities, and has been designated HN. Virions grown in MDBK cells or in the allantoic sac of the chick embryo contain similar amounts of the HN glycoprotein, but differ in their content of the other glycoproteins. Virions grown in MDBK cells contain a large amount of a glycoprotein designated F 0 (MW ~65,000). This glycoprotein is a precursor of a smaller virion glycoprotein, F (MW ~53,000) which is present in only small amount in MDBK cellgrown virions. F 0 can be cleaved to yield F by treatment of virions with trypsin in vitro . Sendai virions grown in the chick embryo lack the precursor protein F 0 , but contain a large amount of F; in this case, proteolytic cleavage of F 0 to yield F occurs in ovo . The Sendai virions grown in MDBK cells, which are deficient in the small glycoprotein F, lack hemolyzing and cell-fusing activities and cannot infect MDBK cells. Virions grown in the chick embryo, which contain much F, possess both these activities and can infect MDBK cells as well as the chick embryo. MDBK cell-grown virions acquire hemolyzing and cell-fusing activities and become infective for MDBK cells when the precursor glycoprotein F 0 is cleaved in vitro to yield F. The results indicate that the small glycoprotein of paramyxoviruses is biologically active and is involved in virus-induced hemolysis, cell fusion, and the initiation of infection. The precise mechanism by which this glycoprotein participates in these reactions remains to be determined, but is now amenable to experimentation. The precursor of this glycoprotein is biologically inactive, but is incorporated into virions grown in some host cells; it may be activated by proteolytic cleavage either in vivo or in vitro . The present results provide a biochemical basis for previously observed host-dependent variation in infectivity, and in hemolysis and cell-fusion induced by paramyxoviruses.

Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the Fl or HA2 viral polypeptides

Abstract A series of oligopeptides have been synthesized with amino acid sequences that resemble those of the N-terminal regions of the paramyxovirus F 1 , polypeptide or the myxovirus HA 2 polypeptide, N-termini generated by proteolytic cleavage which activates infectivity. Oligopeptides with the appropriate structure are highly active, specific inhibitors of the infectivity of each virus, and of cell fusion and hemolysis induced by paramyxoviruses. Structure-activity studies have revealed the following characteristics of the inhibitory activity. Inhibition is amino acid sequence specific, and the presence of the same N-terminal amino acid on the oligopeptide as the viral polypeptide is crucial for activity. Longer peptides are more active than shorter ones with the same initial sequence. The presence of a carbobenzoxy group (Z) on the N-terminal amino acid of the oligopeptide increases inhibitory activity, and the addition of a methyl group to the carboxyl moiety of the C-terminal amino acid decreases activity. The steric configuration of the first two amino acids has a significant effect; optimum activity was obtained with oligopeptides beginning with Z- d -phenylalanine- l -phenylalanine. The results suggest that the oligopeptides competitively interfere with the N-terminal region of the F 1 or HA 2 polypeptides of paramyxoviruses or myxoviruses, respectively. The availability of these oligopeptide inhibitors provides an additional means for determining the exact site and mechanism by which the F 1 and HA 2 polypeptides initiate infection, and for investigating the biochemical and physical events in virus-induced cell fusion and hemolysis, and membrane fusion in general. The finding of specific inhibition of infectivity by oligopeptides which resemble a region of a viral polypeptide also provides a possible new approach to chemical inhibition of viral replication.

Two disulfide-linked polypeptide chains constitute the active F protein of paramyxoviruses.

Abstract The paramyxovirus glycoprotein which is required for virus-induced cell fusion, hemolysis, and the initiation of infection (F protein) has been found with three different viruses to consist of two disulfide-linked glycopolypeptide chains (F1 and F2) which are derived from the precursor glycopolypeptide (F0) by proteolytic cleavage. The larger glycopolypeptide chain (F1) previously identified in SV5, Sendai, and Newcastle disease virions has an estimated molecular weight of 48,000–54,000. The smaller polypeptide chain (F2) has been found to have a molecular weight of ∼10,000–16,000, depending on the virus. The identification of the F2 polypeptide was accomplished by isolating the disulfide-linked complex (F1,2) under nonreducing conditions, followed by reduction of the disulfide bonds and separation of the two polypeptide chains. Although both polypeptides are glycosylated, the F2 polypeptide contains more carbohydrate per unit protein than F1. No free N-terminus could be detected on the F0 or F2 polypeptides of Sendai virus, whereas N-terminal phenylalanine was found on F1. This suggests that the order of the F0 polypeptide is X-NH-F2—Phe-F1—COOH. The finding that with three different paramyxoviruses the biologically active virions possess F1 and F2 polypeptides suggests that this is a general feature of paramyxoviruses, and that the activation of infectivity, cell fusion, and hemolysis is due to a conformational change in the F protein resulting from proteolytic cleavage to form an active complex of two disulfide-linked polypeptide chains.

Isolation of paramyxovirus glycoproteins. Association of both hemagglutinating and neuraminidase activities with the larger SV5 glycoprotein

A method has been developed for the isolation of the glycoproteins of the parainfluenza virus SV5 using the nonionic detergent Triton X-100. Full recovery of hemagglutinating and neuraminidase activities was obtained. By rate zonal centrifugation in sucrose gradients containing 1% Triton X-100 and 0.5 M or 1 M potassium chloride, it was possible to separate the two glycoproteins. Under these conditions, the sedimentation coefficient of the larger glycoprotein, virus protein 2, was 8.9 S and that of the smaller glycoprotein, virus protein 4, was 6.7 S. Each of the proteins aggregated when the detergent and KCl were removed, and the appearance of the aggregates differed with the two proteins. Both hemagglutinating and neuraminidase activities were found to be associated with protein 2; protein 4 exhibited neither activity. The results suggest that in this paramyxovirus both hemagglutinating and neuraminidase activities reside on a single glycoprotein. The biological function of the smaller SV5 glycoprotein remains to be determined.

Reconstitution of membranes with individual paramyxovirus glycoproteins and phospholipid in cholate solution

Abstract A method has been developed for reconstitution of biologically active membranes with individual Sendai virus glycoproteins (HN and F) and phosphatidylcholine. The glycoproteins were isolated in the presence of Triton X-100 and transferred into cholate solution by sedimentation into a sucrose gradient containing 2% cholate. Membranes were then reconstituted with HN or F and phosphatidylcholine by removal of cholate by dialysis. Experiments with radioactively labeled detergents showed that the removal of Triton X-100 by sedimentation into cholate and the removal of cholate by dialysis were essentially complete. With the F protein, vesicles 400–800 A in diameter and filaments 600–800 A in length were formed, depending on the proportions of lipid and protein in the initial mixture. Both structures were covered with glycoprotein spikes. With the HN protein, only vesicles were formed, and the densities of the spikes on the surface was dependent on the initial lipid to protein ratio. Membranes reconstituted from HN protein exhibited hemagglutinating and neuraminidase activities. Reconstituted particles containing the F protein exhibited hemolytic activity when a mechanism was provided to attach the F protein-lipid complex to the cell, i.e., by the addition of wheat germ agglutinin. These results have confirmed the role of the F protein in membrane fusion and have shown that the requirements for F protein activity are the previously demonstrated proteolytic processing of F, the insertion of the F protein into lipid, and the presence of an attachment mechanism.

Protease activation mutants of sendai virus. Activation of biological properties by specific proteases.

Abstract A new class of Sendai virus mutants (pa mutants) is described that exhibit altered specificities with respect to protease activation of infectivity and altered host range. Sendai virus requires proteolytic cleavage of a virion glycoprotein (F 0 to F) in order to be infective. Wild-type virus can be activated in vitro by treatment with trypsin, but not chymotrypsin or elastase, or in vivo by addition of trypsin to cells, e.g., MDBK, which lack activating protease. Mutants have been isolated that are activated by chymotrypsin (pa-c mutants) or elastase (pa-e mutants). Some mutants are no longer activated by trypsin, and these mutants have lost the ability to undergo multiple-cycle replication in the embryonated chick egg unless chymotrypsin or elastase is added to the mutants also activate hemolysis. These findings with pa mutants support the previous conclusion based on results with wild type virus, that a host-dependent cleavage of the F 0 protein is required for the infectivity of Sendai virus and for activation of hemolyzing and cell-fusing activities. The results obtained indicate that the host range and tissue tropism of Sendai virus are determined at least in part by the availability of the appropriate protease required for activation of infectivity.

Immunological studies of the functions of paramyxovirus glycoproteins.

Abstract The HN and F glycoproteins of the paramyxovirus SV5 were purified and monospecific antibodies to each prepared. The effects of bivalent (IgG) and monovalent (Fab) forms of these antibodies on the biological activities of the glycoproteins were determined. Anti-HN antibodies inhibited adsorption of virus to erythrocytes, hemagglutination, and hemolysis. Inhibition of hemolysis was shown to be secondary to the inhibition of adsorption; anti-HN antibodies added after virus adsorption did not affect hemolysis. Anti-HN antibodies inhibited neuraminidase activity, and this inhibition was independent of substrate size in experiments in which virus and antibodies were allowed to react and substrates of different size added, i.e., neuraminlactose and fetuin. This result is in contrast to previous findings with influenza virus in which antibodies inhibited the action of the viral enzyme on the macromolecular substrate only. Thus with SV5, the antibodies appear to inhibit the hydrolytic process directly, rather than sterically hindering the access of the enzyme to large substrates, as in the case in influenza virus. Anti-F antibodies had no effect on virus adsorption or neuraminidase activity, but inhibited hemolysis when added either before or after absorption, confirming the direct involvement of the F protein in the hemolytic process. The inhibition of virus adsorption, neuraminidase, and hemolytic activities by the respective antibodies was accomplished by Fab fragments as well as IgG, indicating that in each case the inhibitory activity was a consequence of a direct effect on individual glycoprotein molecules rather than crosslinking of glycoproteins or aggregation of virions. Both anti-HN and anti-F antibodies neutralized virus infectivity in MDBK and CV-1 cells. Anti-F IgG and Fab, and anti-HN IgG caused essentially complete neutralization in both cell types; however, anti-HN Fab caused only partial neutralization in CV-1 cells.

The relationship of conformational changes in the sendai virus nucleocapsid to proteolytic cleavage of the NP polypeptide

Abstract Previous studies showed that the conformation of the paramyxovirus nucleocapsid varies extensively with regard to the flexibility and coiling of the helix, and changes of this type could be important for virus assembly and transcription of the viral RNA. The helix is tightly coiled and relatively inflexible at high salt concentration (e.g., 1.0 M NaCl) and loosely coiled or extended in low-salt buffer (0.01 M). It has now been found that a salt-induced conformational change can alter the accessibility of the Sendai virus nucleocapsid protein (NP, Mr ∼ 60,000) to cleavage by trypsin. When the nucleocapsid is tightly tightly coiled, NP is cleaved to yield a single major cleavage product (t48, Mr ∼ 48,000) that contains the original N-terminus of the molecule. When the nucleocapsid is in the uncoiled state, t48 is further cleaved to a major N-terminal product, t34, and a second product t15; t15 may be further cleaved to yield t12. Unlike the ∼12,000 daltons of NP that is degraded to small peptides and lost from the nucleocapsid when NP is cleaved to t48, both t34 and t15 (or t12) remain associated with the nucleocapsid structure although thoy are not disulfide bonded to each other. Sendai virus nucleocapsids containing t34 and t15 (or t12) have the same morphological appearance and respond reversibly to changes in salt concentration as do nucleocapsids composed of the native NP or t48. The results indicate that the properties of the NP subunit that are important in determining the helical conformation of the nucleocapsid reside in the N-terminal 48,000 daltons of the molecule and are unaffected by the further cleavages. In contrast to the conformation-dependent exposure of tryptic cleavage sites on NP, the viral RNA in the nucleocapsid is inaccessible to digestion by ribonuclease regardless of salt-dependent changes in conformation, or of the state of tryptic cleavage of NP. Thus neither the tightness of stacking of the helix nor proleolytic cleavages in the NP protein affect the binding of the RNA to NP or its protection from ribonuclease.

Fusion of Sendai virus with liposomes: Dependence on the viral fusion protein (F) and the lipid composition of liposomes☆

The characteristics of fusion of the membrane of Sendai virus with that of liposomes has been investigated using two different methods to monitor the fusion reaction. The first method, which permits quantitation of lipid fused with virus, depends on separation by centrifugation of unfused liposomes from those fused with virus. The second involves the digestion after fusion of internal viral proteins by trypsin contained in liposomes; this assay is completely independent of exchange of lipid between liposomal and viral membranes in the absence of fusion. A fusion-inactive mutant virus, pa-cl, with an uncleaved F protein served as the appropriate control in these experiments. It was found that fusion of the virus with liposomes that contained no protein required cleavage of the F protein; such cleavage was previously shown to be required for fusion of the virus with cell membranes. This indicates the relevance of this model system for studies of fusion. Kinetic studies indicated that at neutral pH fusion was 88% complete in 10 min at 37 degrees. Investigation of the effects of liposomal lipid composition indicated that the presence of cholesterol in the liposomal membrane was required for fusion; a 0.3-0.4-mole fraction of cholesterol was optimal. The presence of neuraminic acid in the membrane was not essential for fusion. The results obtained are compatible with previous evidence suggesting a hydrophobic interaction between the cleaved F protein and the target membrane during fusion.

The hemagglutinating and neuraminidase protein of a paramyxovirus: Interaction with neuraminic acid in affinity chromatography☆

Abstract The two glycoproteins of paramyxoviruses can be separated and purified by affinity chromatography on fetuin which is covalently linked to Sepharose. The glycoproteins are solubilized with Triton X-100 and applied to the column in the Triton-containing solution. The large glycoprotein (HN protein) of Simian virus 5 (SV5) which possesses both hemagglutinating and neuraminidase activities is retained by fetuin-Sepharose at 0° and elutes when the temperature is raised to 25°. The small glycoprotein passes through the column at 0°. This procedure provides a simple and effective means of obtaining high yields of paramyxovirus glycoproteins in pure form. The HN protein can be displaced from the fetuin by the neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid. This indicates that a site on the HN protein binds specifically to the neuraminic acid moieties of the fetuin. This finding is compatible with the hypothesis that the same active site is involved in the neuraminidase and the hemagglutinating activities of the HN protein of paramyxoviruses.