Changes in temperature-sensitivity of haemagglutinin and neuraminidase during adaptation of influenza virus to mice

Temperature-sensitivity of haemagglutinin and neuraminidase of influenza A2 (H2 N2) strains was determined before and after mouse adaptation.Haemagglutinin of all (5) mucoprotein inhibitor sensitive strains and one strain with intermediate properties became more temperature-sensitive. Mucoprotein inhibitor insensitive strains became more resistant.There was no correlation between thermosensitivity of neuraminidase and adaptation to mice.     

Functional and antigenic analyses of the 1918 influenza virus haemagglutinin using a recombinant vaccinia virus expression system

The influenza pandemic of 1918 caused unprecedented levels of morbidity and mortality in its 12-month period of circulation around the globe. The haemagglutinin molecule has been shown to affect the pathogenicity of some subtypes of influenza A viruses. Using a recombinant vaccinia system that allowed expression of the 1918 influenza haemagglutinin, we performed functional assays to assess the glycoprotein's involvement in determining the high pathogenicity of the 1918 virus. We show that in respect of expression levels, proteolytic processing, receptor-binding, membrane fusion and antigenic properties, the haemagglutinin of the 1918 virus is unremarkable when compared with the haemagglutinins of other 'early' H1 influenza viruses. This suggests that whilst the 1918 haemagglutinin, as a new/novel antigen in the human population, was responsible for the influenza pandemic its functions per se were not responsible for the high mortality and acute symptoms experienced by patients infected with the 1918 influenza virus.     

Studies on polysomes synthesizing influenza virus haemagglutinin

Summary A fraction of polysomes synthesizing fowl plague virus (FPV) haemagglutinin (HA) was isolated from an infected chick embryo fibroblast (CEF) culture using a double immunoprecipitation assay. In an immunoprecipitate of HA-synthesizing polysomes (HA precipitate) the content of the HA polypeptide was increased with respect to the M1 + NS1 polypeptides as compared to a preparation of unprecipitated polysomes. In the HA precipitate, besides mRNA coding for HA synthesis, we have detected mRNAs corresponding to genes 1, 2 and 3 coding for high molecular weight P proteins. Studies of a cytoplasmic extract (CE) from FPV-infected CEF cultures in a sucrose density gradient revealed a fraction of polysomes with a sedimentation value of about 500S; the composition of virus-specific polypeptides and mRNA of the fraction was similar to that of the HA precipitate. It is thought that P proteins are synthesized on membrane-bound polysomes located closely to HA-synthesizing polysomes.With 6 Figures     

Contacts between influenza virus N9 neuraminidase and monoclonal antibody NC10

We are interested in studying how influenza virus escapes antibody inhibition. Based on the structure of the complex between N9 NA and monoclonal antibody NC10 Fab (R. L. Malby, W. R. Tulip, V. R. Harley, J. L. McKimm-Breschkin, W. G. Laver, R. G. Webster, and P. M. Colman, 1994, Structure 2, 733-746), we investigated the contribution made by individual amino acids to the stability of the complex. We made conservative changes in residues that are centrally located in the epitope and more drastic changes in peripheral contacts. The mutations made were N200L (removing an N-linked oligosaccharide), N329Q, N345Q, S370T, S372A, N400L, and K432M. Binding of each mutant to NC10 was quantitated by NA inhibition assays and ELISA. Except for N200L and N329Q, the mutants were inhibited by NC10 to the same extent as wild-type NA although with less affinity. The enzyme activity (K(cat)) of N200L is 80% reduced, indicating a defect in folding or assembly; therefore, the loss in binding activity due to the missing sugar residue cannot be assessed. The K(d) for N329Q is sixfold higher than for wild-type NA in the inhibition test, but the same as wild-type in ELISA, indicating a change in disposition of the antibody but no loss of affinity. The results show that the NC10 epitope can accommodate a change at any site and is not dominated by a few high-energy interactions as was found in the NC41 epitope. We propose that the difference lies in the contribution of buried water molecules to the NA-NC10 complex.     

Functional significance of the hemadsorption activity of influenza virus neuraminidase and its alteration in pandemic viruses

Human influenza viruses derive their genes from avian viruses. The neuraminidase (NA) of the avian viruses has, in addition to the catalytic site, a separate sialic acid binding site (hemadsorption site) that is not present in human viruses. The biological significance of the NA hemadsorption activity in avian influenza viruses remained elusive. A sequence database analysis revealed that the NAs of the majority of human H2N2 viruses isolated during the influenza pandemic of 1957 differ from their putative avian precursor by amino acid substitutions in the hemadsorption site. We found that the NA of a representative pandemic virus A/Singapore/1/57 (H2N2) lacks hemadsorption activity and that a single reversion to the avian-virus-like sequence (N367S) restores hemadsorption. Using this hemadsorption-positive NA, we generated three NA variants with substitutions S370L, N400S and W403R that have been found in the hemadsorption site of human H2N2 viruses. Each substitution abolished hemadsorption activity. Although, there was no correlation between hemadsorption activity of the NA variants and their enzymatic activity with respect to monovalent substrates, all four hemadsorption-negative NAs desialylated macromolecular substrates significantly slower than did the hemadsorption-positive counterpart. The NA of the 1918 pandemic virus A/Brevig Mission/1/18 (H1N1) also differed from avian N1 NAs by reduced hemadsorption activity and less efficient hydrolysis of macromolecular substrates. Our data indicate that the hemadsorption site serves to enhance the catalytic efficiency of NA and they suggest that, in addition to changes in the receptor-binding specificity of the hemagglutinin, alterations of the NA are needed for the emergence of pandemic influenza viruses.     

Differential effect of guanidine hydrochloride and heat on the haemagglutinin and neuraminidase of influenza viruses

Summary The effect of guanidine or heat on the haemagglutinin and neuraminidase of thirty strains of influenza virus showed a considerable variation among the strains in the sensitivity of the two surface glycoprotein subunits of the virus to these agents. The kinetics of inactivation was complex, and could not be expressed in terms of a simple mathematical exponential function. However, correlation diagrams plotted with the logarithm of the residual haemagglutinin or neuraminidase activity at a selected point (30 minutes) in the inactivation process showed a general pattern of grouping, which in the case of the haemagglutinin did not completely reflect the conventional serological classification of the strains. The neuraminidase correlation diagram on the other hand evidently reflected the antigenic grouping of the strains into subtypes, N1 and N2, and also differences were detected in the behaviour of strains showing antigenic drift within a subtype. Because guanidine attacks the relatively weaker polar linkages especially hydrogen bonding, its differing effects on the surface glycoproteins of different strains reflect alterations in the molecular configuration of the enzyme molecule. Such changes are also probably associated with antigenic changes such as from one subtype to another (shift) or in antigenic drift.     

Absence of neuraminidase from influenza C virus

Summary Influenza C viruses did not possess neuraminidase activity when examined using either fetuin or sialyllactose as substrate. Purified preparations of influenza C virus inhibited hemagglutination by NWS hemagglutinin. The hemagglutination inhibiting activity was a bolished by treatment of influenza C virus with neuraminidase. These findings indicated the absence of neuraminidase activity on influenza C virus particles.With 2 Figures     

The selective inactivation of influenza virus haemagglutinin by pyridine

Summary Pyridine has been shown to selectively inactivate the haemagglutinating activity of influenza virus. V-antigen and neuraminidase activity were hardly affected and the virions maintained their basic integrity, although some change in morphology was evident. The pyridine treated virus particles obtained were thus unlike the products of virus degradation reported with other solvents or biological agents. Production of multivalent neuraminidase components with no haemagglutinating activity provided the first unequivocal demonstration that neuraminidase does not contribute to the haemagglutinating activity of native virions. The pyridine treated virus retained its antigenic ability although its antigenicity was less than that of the normal virus.     

Intracellular stability of the gene encoding influenza virus haemagglutinin

The intracellular stability of the genome of noninfectious uv-irradiated influenza virus (A/WSN:H1N1) in dividing MDCK cells was investigated using marker rescue techniques. The haemagglutinin gene could still be rescued by infection with A/X49 (H3N2) at 5 weeks postinoculation; its half-life was 13 days.     

Changes in the neuraminidase of neurovirulent influenza virus strains

The influenza virus A/WS/33 has been adapted to mouse brain to produce two neurovirulent derivatives, A/NWS/33 (NWS) and A/WSN/33 (WSN), with the viral neuraminidase gene shown to be the major determinant of neurovirulence. The complete nucleotide sequence of the NA genes from each strain has been determined, which has allowed the identification of changes that have occurred during adaptation to mouse brain. Five changes are shared by the neurovirulent strains. Comparison to the known neuraminidase structure has identified four of these that may affect the active site of the enzyme. In addition, significant differences in the properties of the neuraminidase from the neurovirulent strains were observed relative to the parent strain. While no correlation was observed between neurovirulence and overall neuraminidase activity or preference for a particularN-substitution, the enzymes from both neurovirulent strains showed an increased preference for small substrates and those with 23 linkages, and their activity was potentiated by Ca²⁺ ions.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers L25815-7.     

Aggregation of influenza virus haemagglutinin in the presence of detergents.

In the haemagglutinin (HA) detergent mixtures coexist various protein complexes. Using 125I-HA tracer, gel chromatography, and centrifugation techniques we found protein aggregates comprising up to eight HA trimers. Formation of these structures seemed to be a function of the protein storage medium only. By contrast, special properties of the detergent as well as physicochemical conditions during the protein/detergent interaction had nearly no influence.     

Characterization of temperature sensitive influenza virus mutants defective in neuraminidase

Two temperature sensitive mutants, ts3 and ts11, of the WSN (HON1) strain of influenza virus, belonging to the same recombination group, have a 1000- to 10,000-fold lower infectivity titer and lack hemagglutinating and neuraminidase activity when grown at nonpermissive temperature (39.5°), compared with virus grown at permissive temperature (33°). The patterns of viral polypeptides synthesized in cells infected with these mutants are similar to those found with wild type virus. Neuraminidase activity of the mutant viruses is more temperature labile than the enzyme of the wild type. Despite the low yield, morphologically intact virus particles are found at 39.5°, but in contrast to virus grown at 33° large aggregates of virus accumulate near the cell surface. These aggregated virus particles contain neuraminic acid as demonstrated by a colloidal iron stain. Thus, it is likely that a neuraminic acid containing protein serves as receptor for the hemagglutinin of other virus particles, resulting in the extensive aggregation. Hemagglutinating activity of virus grown at 39.5° is restored by treatment of the aggregates with neuraminidase from Vibrio cholerae. These results suggest that the lack of hemagglutinating activity of mutant virus grown at 39.5° is a consequence of the formation of aggregates of virus particles carrying neuraminic acid on their surface, and that the ts defect is in the neuraminidase but not the hemagglutinin molecule. It is postulated that neuraminidase is essential for the replication of influenza viruses and is required to remove neuraminic acid from the viral envelope to avoid aggregation of the progeny virus.