Three-dimensional structure of an antigenic mutant of the influenza virus haemagglutinin

Antigenic variation in the haemagglutinin (HA) glycoprotein of influenza virus is associated with recurrent epidemics of respiratory disease in man (for review see ref. 1). We have examined the size of structural changes necessary to alter the antigenicity of HA by determining the three-dimensional structure of the HA from an antigenic mutant containing a single amino acid substitution which was selected by growth of virus in the presence of monoclonal antibodies. Here we present evidence that the simple addition of an amino acid side chain which results in only minor local distortions of the structure of the HA is sufficient structural alteration for a virus to escape neutralization by a monoclonal antibody. Our results also demonstrate that single amino acid substitutions can cause only local changes in the HA structure, verifying the assumption made in several studies to locate antigenic sites on the HA and other molecules, and indicate that proposals of large conformational changes to account for variations in HA antigenicity are unnecessary in this case. The structure of the variant antigen has independently been successfully predicted (M. Karplus, personal communication).     

Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA

A synthetic fowl plague virus (FPV) haemagglutinin gene has been cloned in bacteria and the complete sequence of the RNA gene deduced. It is 1,742 nucleotides long and the mRNA codes for 56.3 amino acids in an uninterrupted sequence. The nature of some of the important domains in the haemagglutinin has been established, and their structure is discussed in relation to their function. Extensive amino acid sequence homologies exist between FPV and human influenza haemagglutinins.     

Atomic structure of a human MHC molecule presenting an influenza virus peptide.

Infection by influenza virus results in the stimulation of cytotoxic T lymphocytes specific for killing virally infected cells. Specificity is provided by clonally distributed, hypervariable T-cell receptors on cytotoxic T lymphocytes which react with peptide fragments that are derived from viral proteins expressed in the cytoplasm and 'presented' on the surface of infected cells, bound to class I histocompatibility glycoproteins. Here we describe the structure of the complex between the human class I histocompatibility glycoprotein HLA-Aw68 and the influenza virus nucleoprotein peptide Np 91-99 as determined by X-ray cryocrystallography. Residues at both ends of the peptide are substantially buried in the peptide binding-site, whereas those in the middle of the peptide, P4 to P8, are predominantly exposed and could be recognized directly by T-cell receptors. The extended conformation of the bound viral peptide is remarkably similar to that of a collection of endogenous peptides with a different sequence motif bound to another human allele, HLA-B27. The structure defines in atomic detail the antigenic surface constructed of major histocompatibility complex and viral peptide atoms that is recognized by T-cell receptors.     

Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus

The 'Spanish' influenza pandemic of 1918-19 was the most devastating outbreak of infectious disease in recorded history. At least 20 million people died from their illness, which was characterized by an unusually severe and rapid clinical course. The complete sequencing of several genes of the 1918 influenza virus has made it possible to study the functions of the proteins encoded by these genes in viruses generated by reverse genetics, a technique that permits the generation of infectious viruses entirely from cloned complementary DNA. Thus, to identify properties of the 1918 pandemic influenza A strain that might be related to its extraordinary virulence, viruses were produced containing the viral haemagglutinin (HA) and neuraminidase (NA) genes of the 1918 strain. The HA of this strain supports the pathogenicity of a mouse-adapted virus in this animal. Here we demonstrate that the HA of the 1918 virus confers enhanced pathogenicity in mice to recent human viruses that are otherwise non-pathogenic in this host. Moreover, these highly virulent recombinant viruses expressing the 1918 viral HA could infect the entire lung and induce high levels of macrophage-derived chemokines and cytokines, which resulted in infiltration of inflammatory cells and severe haemorrhage, hallmarks of the illness produced during the original pandemic.     

Increased viral pathogenicity after insertion of a 28S ribosomal RNA sequence into the haemagglutinin gene of an influenza virus

The haemagglutinin glycoprotein HA of influenza viruses is responsible for the attachment of the virus to neuraminic acid-containing receptors at the cell surface and subsequent penetration by triggering fusion of the viral envelope with cellular membranes. To express full activity of the newly synthesized precursor, HA has to be modified by post-translational proteolytic cleavage into the polypeptides HA1 and HA2 by cellular enzymes. If proteases suitable for cleavage are not present in the host cell, the resulting virus particles are non-infectious. During adaptation of the apathogenic influenza virus A/turkey/Oregon/71 to chicken embryo cells, which are not permissive for HA cleavage, we obtained an infectious virus variant with increased pathogenicity. Sequence analysis revealed that during adaptation 54 nucleotides were inserted into the HA gene; their sequence corresponds to a region of the 28S ribosomal RNA. This insertion is probably responsible for increased cleavability of HA, as well as for infectivity and pathogenicity of the adapted virus.     

A distinct bosonic mode in an electron-doped high-transition-temperature superconductor

Despite recent advances in understanding high-transition-temperature (high-T(c)) superconductors, there is no consensus on the origin of the superconducting 'glue': that is, the mediator that binds electrons into superconducting pairs. The main contenders are lattice vibrations (phonons) and spin-excitations, with the additional possibility of pairing without mediators. In conventional superconductors, phonon-mediated pairing was unequivocally established by data from tunnelling experiments. Proponents of phonons as the high-T(c) glue were therefore encouraged by the recent scanning tunnelling microscopy experiments on hole-doped Bi2Sr2CaCu2O8-delta (BSCCO) that reveal an oxygen lattice vibrational mode whose energy is anticorrelated with the superconducting gap energy scale. Here we report high-resolution scanning tunnelling microscopy measurements of the electron-doped high-T(c) superconductor Pr0.88LaCe0.12CuO4 (PLCCO) (T(c) = 24 K) that reveal a bosonic excitation (mode) at energies of 10.5 +/- 2.5 meV. This energy is consistent with both spin-excitations in PLCCO measured by inelastic neutron scattering (resonance mode) and a low-energy acoustic phonon mode, but differs substantially from the oxygen vibrational mode identified in BSCCO. Our analysis of the variation of the local mode energy and intensity with the local gap energy scale indicates an electronic origin of the mode consistent with spin-excitations rather than phonons.     

微波辅助三分子点击化学合成1,4-二取代-1,2,3-三氮唑衍生物

三氮唑是一类重要的有机化合物.本文用点击化学合成了1,4-二取代-1,2,3-三氮唑衍生物.在微波辅助下,碘化亚铜催化叠氮化钠、炔烃、苯磺酸酯三组分反应合成了一系列1,4-二取代-1,2,3-三氮唑衍生物.控制微波功率65～80W,温度50～75℃,反应在15 min内完成,产率81％～94％.新化合物结构用红外、1 ...     

Amino acid sequence changes in the haemagglutinin of A/Hong Kong (H3N2) influenza virus during the period 1968--77

Haemagglutinin molecules from nine strains of A/Hong Kong/68 (H3N2) influenza virus, isolated between 1968 and 1977, were examined for changes in amino acid sequences. At least 18 changes, 9 of which were located precisely, occurred in the soluble tryptic peptides of the large haemagglutinin polypeptide (HA1) during this period. These peptides contained 262 residues (82% of HA1). In HA2, only two changes in 129 residues (58% of HA2) were detected. Sequential changes at a particular locus were not found; and as far as we can tell, once an amino acid changed, it did not change again in any subsequent variant examined.     

Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen.

Immunization with myelin basic protein (MBP) induces experimental allergic encephalomyelitis (EAE), a prototype of CD4+ T-cell mediated autoimmune disease. In rodents, MBP-reactive T-cell clones are specific for a single, dominant determinant on MBP and use a highly restricted number of T-cell receptor genes. Accordingly, EAE has been prevented by various receptor-specific treatments, suggesting similar strategies may be useful for therapy of human autoimmune disease. Here we report that in (SJL x B10.PL)F1 mice, immune dominance of a single determinant, MBP:Ac1-11, is confined to the inductive phase of EAE. In mice with chronic EAE, several additional determinants of MBP in peptides 35-47, 81-100 and 121-140 recall proliferative responses. Most importantly, reactivity to the latter determinants was also detected after induction of EAE with MBP peptide Ac1-11 alone; this demonstrates priming by endogenous MBP determinants. Thus, determinants of MBP that are cryptic after primary immunization can become immunogenic in the course of EAE. Diversification of the autoreactive T-cell repertoire due to 'determinant spreading' has major implications for the pathogenesis of, and the therapeutic approach to, T-cell driven autoimmune disease.     

A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme

In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.     

Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy.

Myotonic dystrophy is the commonest adult form of muscular dystrophy, with an estimated incidence of 1 per 7,500, although this is likely to be an underestimate because of the difficulty of detecting minimally affected individuals. It is a multisystem autosomal dominant disorder of unknown biochemical basis. No case of new mutation has been proven. We have isolated a human genomic clone that detects novel restriction fragments specific to individuals with myotonic dystrophy. A two-allele EcoRI polymorphism is seen in normal individuals, but in most affected individuals one of the normal alleles is replaced by a larger fragment, which varies in length both between unrelated affected individuals and within families. The unstable nature of this region may explain the characteristic variation in severity and age at onset of the disease. A second polymorphism at this locus is in almost complete linkage disequilibrium with myotonic dystrophy, strongly supporting our earlier results which indicated that most cases are descended from one original mutation.