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Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

ABSTRACT: Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to complete fusion. HA is also the most variable, rapidly evolving part of influenza virus, and it is not known whether mechanisms of its activation and fusion are conserved across divergent viral subtypes. Here, we apply hydrogen-deuterium exchange mass spectrometry to compare fusion activation in two subtypes of HA, H1 and H3. Our data reveal subtype-specific behavior in the regions of HA that undergo structural rearrangement during fusion, including the fusion peptide and HA1/HA2 interface. In the presence of an antibody that inhibits the conformational change (FI6v3), we observe that acid-induced dynamic changes near the epitope are dampened, but the degree of protection at the fusion peptide is different for the two subtypes investigated. These results thus provide new insights into variation in the mechanisms of influenza HA's dynamic activation and its inhibition.

SUBMITTER: Garcia NK

PROVIDER: S-EPMC10220487 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Garcia Natalie K NK Kephart Sally M SM Benhaim Mark A MA Matsui Tsutomu T Mileant Alexander A Guttman Miklos M Lee Kelly K KK

The Journal of biological chemistry 20230428 6

Influenza hemagglutinin (HA) is a prototypical class 1 viral entry glycoprotein, responsible for mediating receptor binding and membrane fusion. Structures of its prefusion and postfusion forms, embodying the beginning and endpoints of the fusion pathway, have been extensively characterized. Studies probing HA dynamics during fusion have begun to identify intermediate states along the pathway, enhancing our understanding of how HA becomes activated and traverses its conformational pathway to com ...[more]

PMID: 37121546

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Project description:Influenza virus undergoes rapid evolution by both antigenic shift and antigenic drift. Antibodies, particularly those binding near the receptor-binding site of hemagglutinin (HA) or the neuraminidase (NA) active site, are thought to be the primary defense against influenza infection, and mutations in antibody binding sites can reduce or eliminate antibody binding. The binding of antibodies to their cognate antigens is governed by such biophysical properties of the interacting surfaces as shape, non-polar and polar surface area, and charge.To understand forces shaping evolution of influenza virus, we have examined HA sequences of human influenza A and B viruses, assigning each amino acid values reflecting total accessible surface area, non-polar and polar surface area, and net charge due to the side chain. Changes in each of these values between neighboring sequences were calculated for each residue and mapped onto the crystal structures.Areas of HA showing the highest frequency of pairwise changes agreed well with previously identified antigenic sites in H3 and H1 HAs, and allowed us to propose more detailed antigenic maps and novel antigenic sites for H1 and influenza B HA. Changes in biophysical properties differed between HAs of different subtypes, and between different antigenic sites of the same HA. For H1, statistically significant differences in several biophysical quantities compared to residues lying outside antigenic sites were seen for some antigenic sites but not others. Influenza B antigenic sites all show statistically significant differences in biophysical quantities for all antigenic sites, whereas no statistically significant differences in biophysical quantities were seen for any antigenic site is seen for H3. In many cases, residues previously shown to be under positive selection at the genetic level also undergo rapid change in biophysical properties.The biophysical consequences of amino acid changes introduced by antigenic drift vary from subtype to subtype, and between different antigenic sites. This suggests that the significance of antibody binding in selecting new variants may also be variable for different antigenic sites and influenza subtypes.

Project description:Influenza A H15 viruses are members of a subgroup (H7-H10-H15) of group 2 hemagglutinin (HA) subtypes that include H7N9 and H10N8 viruses that were isolated from humans during 2013. The isolation of avian H15 viruses is, however, quite rare and, until recently, geographically restricted to wild shorebirds and waterfowl in Australia. The HAs of H15 viruses contain an insertion in the 150-loop (loop beginning at position 150) of the receptor-binding site common to this subgroup and a unique insertion in the 260-loop compared to any other subtype. Here, we show that the H15 HA has a high preference for avian receptor analogs by glycan array analyses. The H15 HA crystal structure reveals that it is structurally closest to H7N9 HA, but the head domain of the H15 trimer is wider than all other HAs due to a tilt and opening of the HA1 subunits of the head domain. The extended 150-loop of the H15 HA retains the conserved conformation as in H7 and H10 HAs. Furthermore, the elongated 260-loop increases the exposed HA surface and can contribute to antigenic variation in H15 HAs. Since avian-origin H15 HA viruses have been shown to cause enhanced disease in mammalian models, further characterization and immune surveillance of H15 viruses are warranted.IMPORTANCE In the last 2 decades, an apparent increase has been reported for cases of human infection by emerging avian influenza A virus subtypes, including H7N9 and H10N8 viruses isolated during 2013. H15 is the other member of the subgroup of influenza A virus group 2 hemagglutinins (HAs) that also include H7 and H10. H15 viruses have been restricted to Australia, but recent isolation of H15 viruses in western Siberia suggests that they could be spread more globally via the avian flyways that converge and emanate from this region. Here we report on characterization of the three-dimensional structure and receptor specificity of the H15 hemagglutinin, revealing distinct features and specificities that can aid in global surveillance of such viruses for potential spread and emerging threat to the human population.

Project description:BackgroundH5-subtype highly pathogenic (HP) avian influenza viruses (AIVs) cause high mortality in domestic birds and sporadic infections in humans with a frequently fatal outcome, while H5N1 viruses have pandemic potential. Due to veterinary and public health significance, these HPAIVs, as well as low pathogenicity (LP) H5-subtype AIVs having a propensity to mutate into HP viruses, are under epidemiologic surveillance and must be reported to the World Organization for Animal Health (OIE). Our previous work provided a unique panel of 6 different monoclonal antibodies (mAbs) against H5 hemagglutinin (HA), which meets the demand for high-specificity tools for monitoring AIV infection and vaccination in poultry. In this study, we selected one of these mAbs to develop an epitope-blocking (EB) ELISA for detecting H5 subtype-specific antibodies in chicken sera (H5 EB-ELISA).MethodsIn the H5 EB-ELISA, H5 HA protein produced in a baculovirus-expression vector system was employed as a coating antigen, and the G-7-27-18 mAb was employed as a blocking antibody. The performance characteristics of the assay were evaluated by testing 358 sera from nonimmunized chickens and chickens immunized with AIVs of the H1-H16 subtypes or recombinant H5 HA antigen to obtain the reference and experimental antisera, respectively. The samples were classified as anti-H5 HA positive or negative based on the results of the hemagglutination inhibition (HI) assay, the gold standard in subtype-specific serodiagnosis.ResultsThe H5 EB-ELISA correctly discriminated between the anti-H5 HA negative sera, including those against the non-H5 subtype AIVs, and sera positive for antibodies against the various-origin H5 HAs. Preliminary validation showed 100% analytical and 97.6% diagnostic specificities of the assay and 98.0% and 99.1% diagnostic sensitivities when applied to detect the anti-H5 HA antibodies in the reference and experimental antisera, respectively.ConclusionsThe H5 EB-ELISA performed well in terms of diagnostic estimates. Thus, further optimization and validation work using a larger set of chicken sera and receiver operating characteristic (ROC) analysis are warranted. Moreover, the present assay provides a valuable basis for developing multispecies screening tests for birds or diagnostic tests for humans.

Dataset Information

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

Publications

Structural dynamics reveal subtype-specific activation and inhibition of influenza virus hemagglutinin.

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