X-ray crystallographic determination of the structure of the influenza C virus haemagglutinin-esterase-fusion glycoprotein.

The structure of the haemagglutinin-esterase-fusion (HEF) glycoprotein from influenza C virus has been determined to 3.2 A resolution by X-ray crystallography. A synthetic mercury-containing esterase inhibitor and receptor analogue, 9-acetamidosialic acid alpha-thiomethylmercuryglycoside, was designed as the single isomorphous heavy-atom derivative. The asymmetric unit of one crystal form (form I; P4322, a = b = 155.4, c = 414.4 A) contained an HEF trimer. Six mercury sites identifying the three haemagglutination and three esterase sites were located by difference Patterson map analysis of a 6.5 A resolution derivative data set. These positions defined the molecular threefold-symmetry axis of the HEF trimer. A molecular envelope was defined by averaging a 7.0 A resolution electron-density map, phased by single isomorphous replacement (SIR), about the non-crystallographic threefold-symmetry axis. Iterative non-crystallographic symmetry averaging in real space, solvent flattening and histogram matching were used to extend the phases to 3.5 A resolution. Molecular replacement of the model into a second crystal form (form II; P43212, a = b = 217.4, c = 421.4 A) containing two HEF trimers per asymmetric unit permitted iterative ninefold averaging of the electron density. The 3.5 A electron-density map allowed an unambiguous tracing of the polypeptide chain and identification of N-linked carbohydrates. The model has been refined by least squares to 3.2 A resolution (Rfree = 26.7%).

Structure of the haemagglutinin-esterase-fusion glycoprotein of influenza C virus.

The spike glycoproteins of the lipid-enveloped orthomyxoviruses and paramyxoviruses have three functions: to recognize the receptor on the cell surface, to mediate viral fusion with the cell membrane, and to destroy the receptor. In influenza C virus, a single glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, possesses all three functions. In influenza A and B, the first two activities are mediated by haemagglutinin and the third by a second glycoprotein, neuraminidase. Here we report the crystal structure of the HEF envelope glycoprotein of influenza C virus. We have identified the receptor-binding site and the receptor-destroying enzyme (9-O-acetylesterase) sites, by using receptor analogues. The receptor-binding domain is structurally similar to the sialic acid-binding domain of influenza A haemagglutinin, but binds 9-O-acetylsialic acid. The esterase domain has a structure similar to the esterase from Streptomyces scabies and a brain acetylhydrolase. The receptor domain is inserted into a surface loop of the esterase domain and the esterase domain is inserted into a surface loop of the stem. The stem domain is similar to that of influenza A haemagglutinin, except that the triple-stranded, alpha-helical bundle diverges at both of its ends, and the amino terminus of HEF2, the fusion peptide, is partially exposed. The segregation of HEF's three functions into structurally distinct domains suggests that the entire stem region, including sequences at the amino and carboxy termini of HEF1 which precede the post-translational cleavage site between HEF1 and HEF2, forms an independent fusion domain which is probably derived from an ancestral membrane fusion protein.

Crystallization and preliminary X-ray diffraction studies of the influenza C virus glycoprotein.

Influenza C virus contains a single surface glycoprotein in its lipid envelope which is the hemagglutinin-esterase-fusion glycoprotein (HEF). HEF binds cell-surface receptors, is a receptor-destroying enzyme (a 9-O-acetylesterase), and mediates the fusion of virus and host cell membranes. A bromelain-released soluble form of HEF has been crystallized. Two different tetragonal forms have been identified from crystals with the same morphology [P(1(3))22, a = b = 154.5, c = 414.4 A, and P4(1(3))2(1)2, a = b = 217.4, c = 421.4 A]. Both crystal forms share a common packing scheme. Synchrotron data collection and flash cooling of crystals have been used for high-resolution data collection.

Fusion characteristics of influenza C viruses.

A number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.

Isolation of the influenza C virus glycoprotein in a soluble form by bromelain digestion.

The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.