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      Characterization of H5N1 Influenza Virus Variants with Hemagglutinin Mutations Isolated from Patients

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          ABSTRACT

          A change in viral hemagglutinin (HA) receptor binding specificity from α2,3- to α2,6-linked sialic acid is necessary for highly pathogenic avian influenza (AI) virus subtype H5N1 to become pandemic. However, details of the human-adaptive change in the H5N1 virus remain unknown. Our database search of H5N1 clade 2.2.1 viruses circulating in Egypt identified multiple HA mutations that had been selected in infected patients. Using reverse genetics, we found that increases in both human receptor specificity and the HA pH threshold for membrane fusion were necessary to facilitate replication of the virus variants in human airway epithelia. Furthermore, variants with enhanced replication in human cells had decreased HA stability, apparently to compensate for the changes in viral receptor specificity and membrane fusion activity. Our findings showed that H5N1 viruses could rapidly adapt to growth in the human airway microenvironment by altering their HA properties in infected patients and provided new insights into the human-adaptive mechanisms of AI viruses.

          IMPORTANCE

          Circulation between bird and human hosts may allow H5N1 viruses to acquire amino acid changes that increase fitness for human infections. However, human-adaptive changes in H5N1 viruses have not been adequately investigated. In this study, we found that multiple HA mutations were actually selected in H5N1-infected patients and that H5N1 variants with some of these HA mutations had increased human-type receptor specificity and increased HA membrane fusion activity, both of which are advantageous for viral replication in human airway epithelia. Furthermore, HA mutants selected during viral replication in patients were likely to have less HA stability, apparently as a compensatory mechanism. These results begin to clarify the picture of the H5N1 human-adaptive mechanism.

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          Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.

          J Skehel, D Wiley (2000)
          Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.
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            Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals.

            M Matrosovich, A. Tuzikov, N. Bovin … (2000)
            Interspecies transmission of influenza A viruses circulating in wild aquatic birds occasionally results in influenza outbreaks in mammals, including humans. To identify early changes in the receptor binding properties of the avian virus hemagglutinin (HA) after interspecies transmission and to determine the amino acid substitutions responsible for these alterations, we studied the HAs of the initial isolates from the human pandemics of 1957 (H2N2) and 1968 (H3N2), the European swine epizootic of 1979 (H1N1), and the seal epizootic of 1992 (H3N3), all of which were caused by the introduction of avian virus HAs into these species. The viruses were assayed for their ability to bind the synthetic sialylglycopolymers 3'SL-PAA and 6'SLN-PAA, which contained, respectively, 3'-sialyllactose (the receptor determinant preferentially recognized by avian influenza viruses) and 6'-sialyl(N-acetyllactosamine) (the receptor determinant for human viruses). Avian and seal viruses bound 6'SLN-PAA very weakly, whereas the earliest available human and swine epidemic viruses bound this polymer with a higher affinity. For the H2 and H3 strains, a single mutation, 226Q-->L, increased binding to 6'SLN-PAA, while among H1 swine viruses, the 190E-->D and 225G-->E mutations in the HA appeared important for the increased affinity of the viruses for 6'SLN-PAA. Amino acid substitutions at positions 190 and 225 with respect to the avian virus consensus sequence are also present in H1 human viruses, including those that circulated in 1918, suggesting that substitutions at these positions are important for the generation of H1 human pandemic strains. These results show that the receptor-binding specificity of the HA is altered early after the transmission of an avian virus to humans and pigs and, therefore, may be a prerequisite for the highly effective replication and spread which characterize epidemic strains.
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              Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors.

              Shinya Yamada, Yasuo Suzuki, Takashi Suzuki … (2006)
              H5N1 influenza A viruses have spread to numerous countries in Asia, Europe and Africa, infecting not only large numbers of poultry, but also an increasing number of humans, often with lethal effects. Human and avian influenza A viruses differ in their recognition of host cell receptors: the former preferentially recognize receptors with saccharides terminating in sialic acid-alpha2,6-galactose (SAalpha2,6Gal), whereas the latter prefer those ending in SAalpha2,3Gal (refs 3-6). A conversion from SAalpha2,3Gal to SAalpha2,6Gal recognition is thought to be one of the changes that must occur before avian influenza viruses can replicate efficiently in humans and acquire the potential to cause a pandemic. By identifying mutations in the receptor-binding haemagglutinin (HA) molecule that would enable avian H5N1 viruses to recognize human-type host cell receptors, it may be possible to predict (and thus to increase preparedness for) the emergence of pandemic viruses. Here we show that some H5N1 viruses isolated from humans can bind to both human and avian receptors, in contrast to those isolated from chickens and ducks, which recognize the avian receptors exclusively. Mutations at positions 182 and 192 independently convert the HAs of H5N1 viruses known to recognize the avian receptor to ones that recognize the human receptor. Analysis of the crystal structure of the HA from an H5N1 virus used in our genetic experiments shows that the locations of these amino acids in the HA molecule are compatible with an effect on receptor binding. The amino acid changes that we identify might serve as molecular markers for assessing the pandemic potential of H5N1 field isolates.
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                Author and article information

                Journal
                Journal ID (nlm-ta): mBio
                Journal ID (iso-abbrev): MBio
                Journal ID (hwp): mbio
                Journal ID (pmc): mbio
                Journal ID (publisher-id): mBio
                Title: mBio
                Publisher: American Society of Microbiology (1752 N St., N.W., Washington, DC )
                ISSN (Electronic): 2150-7511
                Publication date (Electronic): 7 April 2015
                Publication date Collection: Mar-Apr 2015
                Volume: 6
                Issue: 2
                Electronic Location Identifier: e00081-15
                Affiliations
                [ a ]Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
                [ b ]Department of Infectious Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
                [ c ]Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
                [ d ]Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
                [ e ]Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt
                [ f ]Health Scientific Hills, College of Life and Health Sciences, Chubu University, Aichi, Japan
                [ g ]Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kagawa, Japan
                [ h ]Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan
                [ i ]Department of Infectious Diseases, Osaka Prefectural Institute of Public Health, Osaka, Japan
                Author notes
                Address correspondence to Yohei Watanabe, nabe@ 123456biken.osaka-u.ac.jp .

                Y.W. and Y. A. contributed equally to this work.

                Editor Terence S. Dermody, Vanderbilt University School of Medicine

                Article
                Publisher ID: mBio00081-15
                DOI: 10.1128/mBio.00081-15
                PMC ID: 4453573
                PubMed ID: 25852160
                SO-VID: 1b8a5c12-51aa-4495-b71a-cb3873536488
                Copyright © Copyright © 2015 Watanabe et al.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 15 January 2015
                Date accepted : 27 February 2015
                Page count
                supplementary-material: 10, Figures: 8, Tables: 1, Equations: 0, References: 49, Pages: 15, Words: 11332
                Categories
                Subject: Research Article
                Custom metadata
                cover-date March/April 2015

                ScienceOpen disciplines: Life sciences
                Data availability:
                ScienceOpen disciplines: Life sciences

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