Wolfgang Garten

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.

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.

Changes in conformation and charge paralleling proteolytic activation of Newcastle disease virus glycoproteins.

Abstract The hemagglutinin-neuraminidase and the fusion glycoprotein of strains Italien and Ulster of Newcastle disease virus were isolated by isoelectric focusing after solubilization with n -octylglucoside or other nondenaturing detergents. The isoelectric points of the glycoproteins varied depending on the virus strain; host-specific variations were not observed. When the precursor F 0 of the fusion protein of strain Ulster was converted by proteolytic cleavage into the complex F 1, 2 , there was a shift in the isoelectric point from pH 6.3 to pH 5.0. When the precursor HN 0 of the same strain was cleaved to HN, its isoelectric point (pH 5.6) did not change. A shift from a more basic to a more acidic pH was also observed, when the isoelectric points of F 0 and F 1, 2 were analyzed under denaturing conditions. These observations are compatible with the concept that F 1 and F 2 are linked in the precursor by a basic intervening sequence that is eliminated in the cleavage reaction. Circular dichroism spectra of the isolated glycoproteins have been determined. The spectra obtained from glycoprotein F 1, 2 of strain Italien and of strain Ulster were similar, but there were strain-specific differences with glycoprotein HN. With the hemagglutinin-neuraminidase and the fusion protein of strain Ulster, precursors and cleavage products showed different spectra. This indicates that proteolytic activation of both glycoproteins is paralleled by a conformational change.

The cleavage site of the hemagglutinin of fowl plague virus

Abstract The amino acid sequence at the cleavage site of the fowl plague virus hemagglutinin has been analyzed. The amino terminus of HA 2 is formed by glycine-343. The carboxy terminus of HA 1 is serine-336 to which occasionally parts of the basic intervening peptide are still attached. The data indicate that the fowl plague virus hemagglutinin like other influenza hemagglutinins is cleaved by the sequential action of a trypsin-like endopeptidase and carboxypeptidase B. There is evidence, however, that the endopeptidases involved in the cleavage of both types of hemagglutinins differ in their specificities.

Haemagglutinin transport mutants

Two mutants (ts1 and ts651) with a temperature sensitive defect in the intracellular transport of the haemagglutinin from the rough endoplasmic reticulum to the plasma membrane have been analysed. Nucleotide sequencing of the haemagglutinin revealed with each mutant two point mutations that are located in the stem region of the molecule.

PROCESSING OF THE HEMAGGLUTININ

SUMMARY Processing of the hemagglutinin involves transport from the rough endoplasmic reticulum to the plasma membrane, glycosylation, and proteolytic cleavage. The tight coupling of these events is demonstrated in experiments in which mutants of fowl plague virus (FPV) with a temperature sensitive defect in transport have been analyzed. Two groups of such mutants have been characterized. With the first group (ts1, ts227), the hemagglutinin is arrested in the rough endoplasmic reticulum. With the second group (ts482, 532, 651), the hemagglutinin migrates to the Golgi apparatus, but does not reach the plasma membrane. The distribution of the carbohydrate side chains on the FPV hemagglutinin has been elucidated. HA 1 has 4 type I side chains attached to asparagine residues 12, 28, 123, and 149. The potential attachment site at asparagine 231 is not glycosylated. HA2 has a type II chain at asparagine 406 and a type I chain at asparagine 478. Comparison with other hemagglutinins demonstrates that the side chains in positions 12, 28, and 478 are conserved. The cleavage site has been analyzed with H3, H7, and H10 hemagglutinins. The available evidence indicates that proteolytic activation involves the action of a trypsin-like protease followed by the action of an exopeptidase of the carboxypeptidase B type: After in vitro cleavage with trypsin, v^iich is paralleled by activation of infectivity, the N-terminus of HA 2 and presumably the C-terminus of HA. are identical to those obtained after in vivo cleavage. This indicates that the carboxypeptidase B activity is present in the virion. After cleavage with the non- activating enzymes thermolysin and chymo trypsin, the cleavage site is shifted by a few amino acids. These observations indicate that activation of infectivity requires a highly specific amino acid sequence at the cleavage site.

Proteolytic Activation of Influenza Viruses: Substrates and Proteases

Like many other viral glycoproteins, the hemagglutinin (HA) of influenza viruses is activated by proteolytic cleavage. Cleavage which is necessary for the fusion activity of the hemagglutinin and thus for the infectivity of the virus is exerted by host cell proteases, and the presence of an appropriate enzyme determines whether infectious virus is made in a given cell. Proteolytic activation is therefore indispensable for effective virus spread in the infected host and has been found to be a prime determinant for virus pathogenicity. This concept has been derived mainly from studies on avian influenza viruses. The pathogenic strains of these viruses are activated by ubiquitous proteases and cause therefore systemic infection mostly leading to rapid death of the animal, whereas activation of the apathogenic strains occurs only in epithelial cells of the respiratory or the enteric tract resulting in local infection of these organs. The mammalian influenza viruses, including the human ones, resemble the apathogenic avian strains in possessing also hemagglutinins of restricted cleavability and in causing usually local infection of the respiratory tract.1