Temperature-sensitive mutants of influenza virus: A mutation in the hemagglutinin gene

A mutant (ts-61S) belonging to a single recombination-complementation group (Group VI) was obtained by segregation of an influenza virus WSN (HON1) temperature-sensitive double mutant (ts-61) that possessed mutational lesions characteristic of Groups V and VI. The segregant retained the thermolabile hemagglutinating activity of the parental mutant, ts-61, but lost the defectiveness in virion RNA synthesis manifested by the parent at the nonpermissive temperature. No hemagglutinating activity developed in cells infected with ts-61S at the nonpermissive temperature. In rescue experiments all HO-serotype progeny from the cross between ts-61S (HO-serotype) and temperature-resistant H3-serotype virus were temperature-sensitive, localizing the ts defect in the hemagglutinin gene. No glycosylated hemagglutinin polypeptide was detected in the polyacrylamide gel electropherogram of cells infected with ts-61S at the nonpermissive temperature, whereas the synthesis of neuraminidase (the other virion glycoprotein) proceeded normally at both permissive and nonpermissive temperatures. The results indicate that the Group VI mutation is in the gene coding for the viral hemagglutinin.     

Design of a recombinant strain of the vaccinia virus containing an expressible gene for influenza A virus hemagglutinin

A recombinant vaccinia virus (VV) strain containing a cloned gene of influenza A/Udorn/307/72 (H3N2) hemagglutinin (HA) gene has been produced. HA expression in CV-1 cells infected with the recombinant virus was determined by enzyme immunoassay. The influenza virus HA titer was 1:64-1:128. When rabbits were inoculated intravenously with the recombinant VaV, antibody titres were 1:5120. The recombinant VaV preparation may be used for generation of monospecific antibody to influenza virus.     

Impact of mutations in the hemagglutinin gene of H5N1 influenza virus on antigenicity and virulence as revealed by site-specific mutagenesis

In our earlier studies, we have shown that amino acid changes in the hemagglutinin (HA) of influenza H5N1 virus escape mutants conferring resistance to monoclonal antibodies (MAbs) may correlate with a decrease of virus virulence for mice and that the virulence can be restored to the initial level by serial passages. In the present study, the mutations identical to those observed in the HA of a low-virulent escape mutant and its readapted variant were introduced into the HA gene by site-specific mutagenesis. The viruses produced by plasmid transfection and containing the HA gene either of A/Vietnam/1203/2004 (H5N1) virus with a deletion at the cleavage site, or of a low-virulent escape mutants, or of its readapted variant, in the presence of 6 genome segments of A/Puerto Rico/8/34 (H1N1) virus and the NA gene of A/Vietnam/1203/2004 (H5N1) virus, were assayed for virulence. Determination of virulence for mice indicated that amino acid substitution in the HA gene of a low-virulent escape mutant produced a decrease of virulence whereas the additional mutation identical to that acquired by the escape mutant in the course of readaptation restored the virulence to initial level. The findings are the first strong evidence for lower H5N1 virus virulence resulting from the amino acid substitution changing the antigenic specificity of HA and for restored virulence arising from compensating mutation in the HA gene.     

Nucleotide sequence of the vaccinia virus hemagglutinin gene

Vaccinia virus hemagglutinin (HA) is expressed at late time of infection cycle, and it is nonessential for virus growth. Location of the HA structural gene was determined by hybrid-arrested and hybrid-selected translation methods at the right terminus of the HindIII A fragment. The position of the HA gene was confirmed by the production of the complete HA protein in the cells transfected with the plasmid containing that region. Examination of this nucleotide sequence revealed the positions of cleavage sites for a number of restriction endonucleases. The deduced amino acid sequence revealed that the HA protein is a member of typical surface membrane glycoproteins. Comparison of the nucleotide sequence upstream of the HA coding region with corresponding region of other late genes suggested the existence of the consensus decanucleotides TTCATTTa/tGT between 34 to 18 bp upstream to the initiation codon followed by a cluster of A or T, a unique feature of the late genes of vaccinia virus. These results in conjunction with the ease of isolating HA- mutants provide a basis for a new site suitable for inserting foreign genes.     

Distribution of hemagglutinin and neuraminidase on influenza virions as revealed by immunoelectron microscopy

Monoclonal antibodies specific for the hemagglutinin (HA) or the neuraminidase (NA) of influenza viruses were used in immunoelectron microscopic studies to determine the distribution of the two surface spikes on the virion. Indirect immunogold staining revealed that the HA is uniformly distributed on the virion while the NA occurs in discrete areas. Crosslinking and low temperature studies argue against redistribution of the HA and NA after antibody attachment and indicate that the NA on influenza virus occurs in patches.     

Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.     

Mutations in the hemagglutinin gene associated with influenza virus resistance to norakin

Summary The nucleotide sequences of the hemagglutinin genes of four norakinresistant mutants of Influenza A/FPV/Weybridge were determined and compared to the wild-type hemagglutinin. All mutants show one or two amino acid substitutions which are discussed to destabilise the pH 7 conformation of hemagglutinin.     

Gene and protein sequence of an influenza neuraminidase with hemagglutinin activity

An influenza virus neuraminidase (NA) of the N9 subtype also has hemagglutinin (HA) activity (W. G. Laver, P. M. Colman, R. G. Webster, V. S. Hinshaw, and G. M. Air (1984), Virology 137, 314-323). To determine sequence relationships between this NA and other known NA and HA subtype sequences, and as a necessary step toward a complete structure determination, we have cloned a full-length copy of the coding sequence of the N9 NA of influenza virus A/tern/Australia/G70C/75 into the plasmid pUC9 using SalI linkers. The gene was sequenced by directed subcloning into the single-stranded phage vectors M13mp19 and M13mp18 and use of the dideoxy procedure. Most of the NA sequence was also obtained by direct protein sequencing of tryptic peptides. The N9 NA has 43 and 44% homology when compared to N1 or N2 sequences, respectively. There is no significant homology to any known HA sequence, or to the HN protein of the paramyxovirus SV5. Like the other NA molecules, the N9 NA is anchored in the membrane by an N-terminal hydrophobic region, from which biologically active heads can be released by pronase.     

Changes in the hemagglutinin gene of the neurovirulent influenza virus strain A/NWS/33

The neurovirulent strain of influenza A virus, A/NWS/33, is able to infect a large range of cell types, including mouse brain cells, which are not infected by its parent, A/WS/33. This seems to be largely due to the hemagglutinin of A/NWS/33. The complete nucleotide sequence of the HA genes of both strains has been determined and a comparison revealed a number of changes. Analysis showed that the virulence capabilities of the NWS HA involve at least three different mechanisms: (a) loss of a glycosylation site; (b) a change at the cleavage site; and (c) a substitution in HA₂, which may increase the pH of fusion.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers U08903-4.     

Extragenic suppression of temperature-sensitive mutations in RNA segment 8 by replacement of different RNA segments with those of other influenza A virus prototype strains

Extragenic suppression by replacement of different RNA segments has been detected during rescue by other prototype influenza strains of temperature-sensitive (ts) mutants of an influenza virus with lesions in segment 8. The ts⁺ recombinants obtained in this way usually had not replaced the RNA segment (= gene) with the is lesion, but they had replaced other RNA segments depending on the strain used for rescue. Backcross experiments with the parental strain showed that the is lesion was extragenically suppressed by introducing a foreign RNA segment. The results are discussed in terms of a cooperation between one of the gene products of RNA segment 8 (nonstructural proteins) and the RNA polymerase complex.     

Consolidation of intramolecular disulfide bonds in influenza virus hemagglutinin as an element of intracellular maturation

Electrophoretic behavior of influenza virus hemagglutinin during SDS electrophoresis in polyacrylamide gel is critically dependent on the life time in the infected cells and also on the conditions of sample preparation and analysis. During electrophoresis of total cell lysate proteins under nonreducing conditions the short-labeled hemagglutinin is detected as multiple bands, electrophoretic mobility of most of them being lower than that of hemagglutinin of viral particles. This heterogeneity failed to be detected during electrophoresis under reducing conditions which is indicative of the differences in the number or direction of intramolecular disulfide bonds between short-labeled and mature hemagglutinin molecules. After chasing at 37 or 20 degrees hemagglutinin gradually assumes an electrophoretic character identical to that of virion protein. Chasing at 0 degrees or the substitution of parafluorophenyl alanine for phenylalanine in the maintenance medium during labeling prevents maturation. At the same time, both iodacetamide perfusion of infected cells and the preparation of nuclei-free extract prior to SDS lysis result in a marked increase in the yield of disulfide mature short-labeled hemagglutinin. These results suggest that disulfide maturation in hemagglutinin proceeds in two stages: a relatively rapid (with respect to synthesis completion) formation of intramolecular disulfide bonds as such followed by a much slower consolidation of bridges against the action of endogenous cell reductants which activate during lysis. Consolidation may be caused by two factors: trimerization of hemagglutinin monomers or their covalent post-translational modifications.     

Transgenic mice that express different forms of the influenza virus hemagglutinin as a neo-self-antigen

We have generated transgenic mouse lineages that express the influenza virus hemagglutinin in different physical forms. One kind expresses the full-length hemagglutinin molecule as a cell surface glycoprotein and can be recognized by hemagglutinin-specific B and T cells. The other expresses a truncated polypeptide corresponding to the N-terminal third of the hemagglutinin molecule. This polypeptide encodes known hemagglutinin-specific T-cell determinants; however, it contains no native B-cell epitopes, since these depend on the conformation of the fully folded protein. In each case, the hemagglutinin transgenic mice display ubiquitous expression of transgenic messenger RNA and induce T-cell tolerance to the transgene-encoded T-cell determinant site 1. Thus, the hemagglutinin is a neo-self-antigen in both kinds of hemagglutinin transgenic mice and should provide a useful system for understanding the factors and mechanisms that govern tolerance and autoimmunity to self-antigens.     

Isolation of preparative amounts of influenza virus hemagglutinin by an affinity chromatographic method

The use of affinity column sepharose-tyrosine-sulfanilic acid permits one to obtain preparative amounts of pure hemagglutinin of the types H1, H2, H3 from the total amount of surface glycoproteins solubilized by octylglycoside from antigenically different strains of influenza A virus. The yield of hemagglutinin ranges from 30% to 84% depending on the virus strain.     

Protection against influenza virus infection by intranasal administration of hemagglutinin vaccine with chitin microparticles as an adjuvant

Chitin in the form of microparticles (chitin microparticles, CMP) has been demonstrated to be a potent stimulator of macrophages, promoting T-helper-1 (Th1) activation and cytokine response. In order to examine the mucosal adjuvant effect of CMP co-administered with influenza hemagglutinin (HA) vaccine against influenza infection, CMP were intranasally co-administered with influenza HA vaccine prepared from PR8 (H1N1) virus. Inoculation of the vaccine with CMP induced primary and secondary anti-HA IgA responses in the nasal wash and anti-HA IgG responses in the serum, which were significantly higher than those of nasal vaccination without CMP, and provided a complete protection against a homologous influenza virus challenge in the nasal infection influenza model. In addition, CMP-based immunization using A/Yamagata (H1N1) and A/Guizhou (H3N2) induced PR8 HA-reactive IgA in the nasal washes and specific-IgG in the serum. The immunization with A/Yamagata and CMP resulted in complete protection against a PR8 (H1N1) challenge in A/Yamagata (H1N1)-vaccinated mice, while that with A/Guizhou (H3N2) and CMP exhibited a 100-fold reduction of nasal virus titer, demonstrating the cross-protective effect of CMP and influenza vaccine. It is suggested that CMP provide a safe and effective adjuvant for nasal vaccination with inactivated influenza vaccine.     

Cleavage of influenza virus hemagglutinin as affected by serum plasmin in cell culture and in vivo

The effect of chicken, canine, and porcine plasm containing plasmin proenzyme, plasminogen, on influenza virus hemagglutinin produced in homologous and heterologous tissue cells was studied. The cells incubated with the homologous plasm were found to produce virions containing both cleaved and uncleaved hemagglutinin whereas the cells incubated without plasm or with heterologous plasm produced virions with uncleaved hemagglutinin. The infectious activity of the virus produced by cells with the homologous plasm was much higher than that of the virus grown without the latter or with heterologous plasm. The addition to the culture medium of plasminogen inhibitors together with plasm eliminated the proteolytic effect of the plasm on virion hemagglutinins resulting in the production of virions with uncleaved hemagglutinin and low infectious activity. In vivo, in experimental influenza infection of mice and chickens, highly infectious virus with cleaved hemagglutinin was isolated from the organs of the animals. The organs of the animals inoculated with inhibitors of proteolytic enzymes yielded virus of low infectivity with uncleaved hemagglutinin. Administration of proteases inhibitors to the infected animals prevented the spread of virus infection in animals and had a therapeutic effect. The experimental data suggest that activation of virions with proteolytic enzymes of the host, in particular, plasmin by means of hemagglutinin cleavage is the key mechanism in the development and spread of influenza infection in the host.     

Role of influenza A virus hemagglutinin in neurovirulence for mammalians

Influenza viruses with potential neuroinvasiveness for humans emerged in Hong Kong in 1997. Prophylactic and therapeutic strategies are urgently needed for controlling the central nervous system complications caused by influenza. Here we review recent advances toward understanding of the possible mechanisms of the neuropathogenesis of influenza virus infection, especially focusing on the role of viral hemagglutinin glycoprotein.     

Evolution of the hemagglutinin gene of H3N8 canine influenza virus in dogs

With the widespread use of a recently developed canine influenza virus (CIV) H3N8 vaccine, continual molecular evaluation of circulating CIVs is necessary for monitoring antigenic drift. The aim of this project was to further describe the genetic evolution of CIV, as well as determine any genetic variation within potential antigenic regions that might result in antigenic drift. To this end, the hemagglutinin gene of 19 CIV isolates from dogs residing in Colorado, New York, and South Carolina humane shelters was sequenced and compared to CIV strains isolated during 2003–2012. Phylogenetic analysis suggests that CIV might be diverging into two geographically distinct lineages. Using a mixed-effects model for evolution and single likelihood ancestor counting methods, several amino acid sites were found to be undergoing selection pressure. Additionally, a total of six amino acid changes were observed in two possible antigenic sites for CIVs isolated from Colorado and New York humane shelters between 2009 and 2011. As CIV isolates might be diverging into geographically distinct lineages, further experiments are warranted to determine the extent of antigenic drift occurring within circulating CIV.