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Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus.

ABSTRACT: Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in The Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates from the same outbreak. A mutation which introduces a potential glycosylation site at Asn123 in the NL219 hemagglutinin was postulated to contribute to the pathogenic properties of this virus. To study this further, we have expressed the NL219 hemagglutinin in a baculovirus expression system and performed a structural analysis of the hemagglutinin in complex with avian and human receptor analogs. Glycan microarray and kinetic analysis were performed to compare the receptor binding profile of the wild-type recombinant NL219 HA to a variant with a threonine-to-alanine mutation at position 125, resulting in loss of the glycosylation site at Asn123. The results suggest that the additional glycosylation sequon increases binding affinity to avian-type ?2-3-linked sialosides rather than switching to a human-like receptor specificity and highlight the mechanistic diversity of these pathogens, which calls attention to the need for further studies to fully understand the unique properties of these viruses.

SUBMITTER: Yang H

PROVIDER: S-EPMC3421765 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus.

Yang Hua H Carney Paul J PJ Donis Ruben O RO Stevens James J

Journal of virology 20120606 16

Recurrence of highly pathogenic avian influenza (HPAI) virus subtype H7 in poultry continues to be a public health concern. In 2003, an HPAI H7N7 outbreak in The Netherlands infected 89 people in close contact with affected poultry and resulted in one fatal case. In previous studies, the virus isolated from this fatal case, A/Netherlands/219/2003 (NL219) caused a lethal infection in mouse models and had increased replication efficiency and a broader tissue distribution than nonlethal isolates fr ...[more]

PMID: 22674977

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Project description:The influenza A virus is of global concern for the poultry industry, especially the H5 and H7 subtypes as they have the potential to become highly pathogenic for poultry. In this study, the hemagglutinin (HA) of a low pathogenic avian influenza virus of the H7N7 subtype isolated from a Swedish mallard Anas platyrhynchos was sequenced, characterized and transiently expressed in Nicotiana benthamiana. Recently, plant expression systems have gained interest as an alternative for the production of vaccine antigens. To examine the possibility of expressing the HA protein in N. benthamiana, a cDNA fragment encoding the HA gene was synthesized de novo, modified with a Kozak sequence, a PR1a signal peptide, a C-terminal hexahistidine (6×His) tag, and an endoplasmic retention signal (SEKDEL). The construct was cloned into a Cowpea mosaic virus (CPMV)-based vector (pEAQ-HT) and the resulting pEAQ-HT-HA plasmid, along with a vector (pJL3:p19) containing the viral gene-silencing suppressor p19 from Tomato bushy stunt virus, was agro-infiltrated into N. benthamiana. The highest gene expression of recombinant plant-produced, uncleaved HA (rHA0), as measured by quantitative real-time PCR was detected at 6 days post infiltration (dpi). Guided by the gene expression profile, rHA0 protein was extracted at 6 dpi and subsequently purified utilizing the 6×His tag and immobilized metal ion adsorption chromatography. The yield was 0.2 g purified protein per kg fresh weight of leaves. Further molecular characterizations showed that the purified rHA0 protein was N-glycosylated and its identity confirmed by liquid chromatography-tandem mass spectrometry. In addition, the purified rHA0 exhibited hemagglutination and hemagglutination inhibition activity indicating that the rHA0 shares structural and functional properties with native HA protein of H7 influenza virus. Our results indicate that rHA0 maintained its native antigenicity and specificity, providing a good source of vaccine antigen to induce immune response in poultry species.

Project description:UnlabelledHighly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3'SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLe(c)) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLe(x)), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors.ImportanceHere, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor-binding site of HA which appeared in HA of LPAIVs in the course of transmission of LPAIVs from wild waterfowl into poultry flocks, with subsequent adaptation in poultry. The identification of LPAIVs with receptor characteristics of HPAIVs argues that the sialic acid-binding specificity of the HA may be used as a novel phenotypic marker of HPAIVs.

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Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus.

Publications

Structure and receptor complexes of the hemagglutinin from a highly pathogenic H7N7 influenza virus.

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