Hans-Dieter Klenk

Activation of influenza A viruses by trypsin treatment

Abstract A comparative analysis has been carried out on the infectivity of virus of several influenza A strains grown in different host systems. Strains A/swine/Shope/31 (Hsw1N1), A/PR/8/34 (HON1), A/FM/1 (H1N1), A/Singapore/1/57 (H2N2), A/equine/Miami/1/63 (Heq2Neq2), and A/chick/Germany/49 (Hav2Neq1) exhibit host-dependent differences in infectivity. Virions grown in embryonated eggs and cultures of chorioallantoic membrane cells are highly infectious, whereas virions grown in cultures of chick embryo cells have a low infectivity that significantly increases after treatment in vitro with trypsin. In contrast, fowl plague viruses do not show host-dependent variations in infectivity. Virions grown in all host systems tested are highly infectious, and the infectivity of virions grown in chick embryo cells cannot be enhanced by trypsin treatment. The activation of virus particles appears to be based on the cleavage of hemagglutinin glycoprotein HA. This concept is supported by the following observations: (i) In virions of low infectivity only uncleaved glycoprotein HA can be detected. Virions of high infectivity exhibit complete or at least partial cleavage of the hemagglutinin. (ii) The activation of virions by trypsin treatment is always paralleled by cleavage of HA. (iii) Cleavage of HA is the only effect which can be detected after trypsin treatment. The neuraminidase is neither inactivated nor removed from the virion. (iv) Studies on recombinants of virus N and fowl plague virus (Rostock) show that host-dependent variation of infectivity and activation by trypsin, features specific for parent virus N, are found only with recombinant N(H)-FPV/Ro(N) but not with recombinant FPV/Ro(H)-N(N). Efficient plaque formation and serial passages are possible only if highly infectious particles are formed in a given host system. Thus, all strains analyzed undergo, in the absence of trypsin, successive growth cycles in eggs and chorioallantoic membrane cells and form plaques in chorioallantoic membrane cells. In contrast, in chick embryo cells only viruses containing the fowl plague virus hemagglutinin produce plaques and replicate under multiple cycle conditions without the addition of trypsin. The data show that cleavage of HA is not a precondition for virus assembly and hemagglutinating activity, but that it is necessary for infectivity. These findings are compatible with the hypothesis that, in addition to its role in adsorption, the hemagglutinin has another function in the infection process and cleavage is required for this function.

Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus.

Abstract In search of a molecular basis underlying the variations in virulence observed with different strains of Newcastle disease virus, a comparative study has been carried out on the biosynthesis and function of the viral glycoproteins. Five virulent (Italien, Herts, Field Pheasant, Texas, Warwick) and five avirulent strains (La Sota, B 1 , F, Queensland, Ulster) have been analyzed. They were grown in five different host systems (embryonated eggs, cultures of BHK21-F, MDBK, chick embryo, and chick chorioallantoic membrane cells). Glycoprotein F (MW 56,000) which is responsible for hemolysis and cell fusion has been found with all strains to be derived by proteolytic cleavage from the precursor glycoprotein F o (MW 68,000). With strains Queensland and Ulster, in addition, a precursor glycoprotein HN o (MW 82,000) has been identified which is converted, again by proteolytic cleavage, into the hemagglutinin-neuraminidase glycoprotein HN (MW 74,000). Cleavage of F o is necessary for the expression of cell fusing and hemolytic activity, and the available evidence suggests that cleavage of HN o is paralleled by an enhancement of hemagglutinating and neuraminidase activity. However, activation of the glycoproteins is not required for virus assembly. Thus, virus particles containing the precursor F o may be formed which have a reduced infectivity. Infectivity is even lower, if both glycoproteins are present in the uncleaved form. After in vitro treatment with trypsin, such particles display full biological activity. Whether the glycoproteins are cleaved in vivo depends on the virus strain and on the host cell. With virulent strains, cleavage occurs in all host systems analyzed, and the virions formed contain HN and F. With avirulent strains, however, this is the case only in the embryonated egg and in cultures of chorioallantoic membrane cells. All other cells produce particles containing uncleaved glycoproteins. From these observations the following conclusions can be drawn: only a few host systems are permissive for avirulent strains, i.e., they produce highly infectious virus; other systems are nonpermissive for these strains, i.e., they produce defective virus; in contrast, all host systems studied are permissive for virulent strains. This concept is supported by the finding that multiple replication cycles and plaque formation occur only in permissive cells or in a nonpermissive culture after substitution of trypsin. Thus, plaque assays are now available for avirulent strains, either by the use of MDBK and chick embryo cells in the presence of trypsin or by the use of chorioallantoic membrane cells. These observations demonstrate striking differences in host range between virulent and avirulent strains which are determined by the susceptibility of the envelope glycoproteins to proteolytic cleavage. It is suggested that these differences account at least in part for the variations in the virulence of Newcastle disease virus.

Proteolytic cleavage of influenza virus hemagglutinins: primary structure of the connecting peptide between HA1 and HA2 determines proteolytic cleavability and pathogenicity of avian influenza viruses

The structural basis for the different proteolytic cleavability of influenza virus hemagglutinin (HA) was investigated with a group of pathogenic and nonpathogenic avian influenza viruses belonging to the antigenic subtype H7 (Hav1). Infected cel lysates or lystates of purified virus particles were subjected to two-dimensional gel electrophoresis. The first dimension, isoelectric focusing,- was done under nonreducing conditions, the second dimension, SDS-PAGE, under reducing conditions. The results obtained permit the following conclusions: The amino acid sequence of the connecting peptide between HA1 and HA2 determines proteolytic cleavability by a trypsin-like cellular enzyme. Upon proteolytic cleavage of HA of pathogenic strains, peptides of differing positive charge were eliminated. These HAs have, however, significantly more basic connecting peptides than HAs of nonpathogenic viruses. HAs of nonpathogenic H7 strains appear to have a connecting peptide similar to the human influenza viruses, since treatment of these viruses with trypsin results in a similar small charge shift which probably corresponds to the elimination of one basic amino acid. Thus, the primary structure of the connecting peptide determines biological activation and thereby pathogenicity of these viruses.

The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses

Abstract Comparative studies on naturally occurring avian influenza viruses have been carried out in order to investigate the determinant(s) for pathogenicity for chickens. At least one virus isolate from each of the nine different hemagglutinin (HA) subtypes was included. The polypeptides of these viruses were studied by analyzing infected cell extracts on SDS-polyacrylamide gels. Both viral glycoproteins, HA and neuraminidase, showed remarkable variation in their electrophoretic mobility even among serologically closely related viruses. Pulse-chase experiments revealed that most avian influenza virus strains had an HA which was not susceptible to proteolytic cleavage in MDCK, turkey (TEC), and chicken embryo cells (CEC). Only viruses belonging to the subtype Hav5 and some strains of the subtype Hav1 possessed a cleaved HA in these cells. Only the virus strains with cleaved HA were produced in infectious form in MDCK, CEC, TEC, as well as in duck embryo cells (DEC) and quail embryo cells (QEC). The other virus strains produced plaques in these cells only in the presence of trypsin. There was a strict correlation between the cleavability of the HA, the potential of the virus to be produced in infectious form in a wide range of host cells, and their pathogenicity for chickens. No evidence was obtained for an involvement of the neuraminidase in determining pathogenicity. For the nonpathogenic viruses it could be shown that they can replicate and produce infectious progeny in some organs of the chicken. The results obtained permit the conclusion that in naturally occurring avian influenza viruses the structure of the hemagglutinin, that is its susceptibility to proteolytic cleavage in a broad spectrum of host cells, is the determining factor for pathogenicity.

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN0 and F0, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN0 and F0 together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN0 was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN0 and F0 with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN0 can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F0 shows a specific requirement for trypsin.

Inhibition of glycoprotein biosynthesis of influenza virus by d-glucosamine and 2-deoxy-d-glucose

Abstract Chick embryo fibroblasts infected with fowl plague virus contain, in addition to six structural proteins, two nonstructural proteins, one of which is a precursor glycoprotein and yields after cleavage the two structural glycoproteins of the hemagglutinin. High concentrations of d -glucosamine and 2-deoxy- d -glucose inhibit the formation of hemagglutinin, neuraminidase, and mature virions. Analysis by polyacrylamide gel electrophoresis reveals that under these conditions the virus-specific carbohydrate-free polypeptides are still being formed. However, all viral glycoproteins are missing. Instead of the glycoproteins a single polypeptide can be detected. The available evidence suggests that this protein is the carbohydrate-free polypeptide chain of the glycoprotein precursor of the hemagglutinin and that it accumulates in infected cells when glycosylation is blocked.

Proteolytic activation of the influenza virus hemagglutinin: The structure of the cleavage site and the enzymes involved in cleavage

The cleavage site of the hemagglutinin of influenza strains A/chick/Germany/49 (H10), A/Port Chalmers/1/73 (H3), A/Victoria/3/75 (H3), and A/WSN/33 (H1) has been analyzed after in vivo activation and after in vitro activation with trypsin or other proteases of trypsin-like specificity. Sequence analyses revealed that the amino termini of HA2 and the carboxy termini of HA1 are identical with in vitro- and in vivo-activated hemagglutinin. Proteolytic activation is paralleled by an acidic shift in the isoelectric point of the hemagglutinin, reflecting the elimination of an intervening arginine residue at the cleavage site. These data indicate that two enzymes are involved in the activation of the hemagglutinin: after the initial action of trypsin or a trypsin-like endoprotease furnished by the host, an exopeptidase of the carboxypeptidase B type appears to remove the arginine from the cleavage site. The observation that arginine is eliminated, when the hemagglutinin is cleaved in vitro with trypsin as the only enzyme added to purified virus, indicates that the carboxypeptidase B is a constituent of the virus particle. The available evidence indicates that there are similarities in the activation mechanism between the influenza virus hemagglutinin and a series of prohormones and proenzymes. The arginine is not removed when the hemagglutinin is cleaved by the nonactivating enzymes chymotrypsin and thermolysin. If compared to the cleavage site of trypsin, the cleavage site of chymotrypsin is shifted in carboxy-terminal direction by three amino acids and that of thermolysin by only one amino acid. These observations further substantiate the involvement of a carboxypeptidase B in activation. In addition, they support the concept that activation of the hemagglutinin requires a high structural specificity at the cleavage site.

Studies on the assembly of the envelope of Newcastle disease virus

Abstract The association of the envelope proteins of Newcastle disease virus with membranes of infected BHK 21-F cells and their incorporation into mature envelopes has been investigated in a study employing cell fractionation. The principal fractions obtained by sucrose density gradient centrifugation of cytoplasmic extracts were rough endoplasmic reticulum and smooth membranes derived predominantly from smooth endoplasmic reticulum and Golgi apparatus. Furthermore, by adsorption to red blood cells it was possible to isolate virions and a hemadsorptive fraction of smooth membranes believed to be immediate precursors of mature envelopes. In addition to the cytoplasmic fractions, plasma membranes obtained as cell ghosts have been analyzed. Each fraction showed a distinct pattern of virus-specific proteins. Pulse-chase experiments indicated that glycoprotein HN and Fo were synthesized on the rough endoplasmic reticulum and transferred from there via smooth intracellular membranes to the plasma membrane and into virions. In the course of migration, Fo is converted to F. In contrast to the glycoproteins, protein M was found to be incorporated into the plasma membrane immediately after synthesis. Pulse-chase experiments also demonstrated that this protein appears in the hemagglutinating fraction of smooth membranes and in mature virions more rapidly than the glycoproteins. These results suggest that M is incorporated into membranes that contain already viral glycoproteins and that this process is one of the last steps in envelope assembly.

On the structure of the influenza virus envelope

The polypeptide, lipid, and carbohydrate constituents of intact fowl plague virus and of subviral particles have been determined. The protein pattern of the intact virion consists of 3 carbohydrate-free polypeptides and of 3 glycoproteins with a total molecular weight of 296,000. Isolated spikes contain only these 3 glycoproteins. When the spikes are removed from the virion by protease degradation, a subviral particle can be recovered which has lost the glycoproteins but still contains the carbohydrate-free polypeptides and the entire lipid. Intact virions can be precipitated by concanavalin A. The carbohydrate receptor specific for this phytagglutinin is located on the viral spikes. Phytagglutinin from Dolichos biflorus precipitates only particles devoid of spikes, because its glycolipid receptor is not accessible unless the spikes are removed from the virion. These findings indicate that carbohydrates are present in the viral envelope in 2 layers: (1) in the spikes covalently linked to protein, (2) in the lipid layer covalently linked to lipid. The serological specificities of the protein- and the lipid-bound carbohydrates are different.

Fusion between cell membrane and liposomes containing the glycoproteins of influenza virus

Abstract Glycoproteins of influenza virus strains were incorporated into liposomes by a dialysis procedure, using octylglucoside as detergent. Liposomes containing either the cleaved or uncleaved hemagglutinin of the virus were tested for fusion activity with cellular membranes. Electron microscopic examination as well as microinjection studies revealed that liposomes containing the cleaved hemagglutinin could fuse with cell membranes. In contrast, liposomes containing the uncleaved hemagglutinin were merely adsorbed to the cell surface and fusion occurred only after treatment with trypsin. Native virus particles with the cleaved hemagglutinin could be shown to fuse with liposomes containing cellular receptors of influenza virus. From these results and the known correlation existing between cleavage of hemagglutinin and infectivity of influenza virus, it is suggested that fusion may be an important step in penetration of the nucleocapsid of influenza virus into host cells.