Conceived and designed the experiments: CM LR MC. Performed the experiments: CM. Analyzed the data: CM MS. Contributed reagents/materials/analysis tools: MW. Wrote the paper: CM LR MC.
The authors have declared that no competing interests exist.
In influenza, the envelope protein hemagglutinin (HA) plays a critical role in viral entry by first binding to sialic acid receptors on the cell surface and subsequently mediating fusion of the viral and target membranes. In this work, the receptor binding properties of influenza A HA from different subtypes (H1 A/California/04/09, H5 A/Vietnam/1205/04, H5 A/bar-headed goose/Qinghai/1A/05, and H9 A/Hong Kong/1073/99) have been characterized by NMR spectroscopy. Using saturation transfer difference (STD) NMR, we find that all HAs bind to the receptor analogs 2,3-sialyllactose and 2,6-sialyllactose, with subtle differences in the binding mode. Using competition STD NMR, we determine the receptor preferences for the HA subtypes. We find that H5-Qinghai and H9-Hong Kong HA bind to both receptor analogs with similar affinity. On the other hand, H1 exhibits a clear preference for 2,6-sialyllactose while H5-Vietnam exhibits a clear preference for 2,3-sialyllactose. Together, these results are interpreted within the context of differences in both the amino acid sequence and structures of HA from the different subtypes in determining receptor preference.
The membrane glycoproteins hemagglutinin (HA) and neuraminidase (NA) play critical roles in influenza infection
HA, as well as analogous envelope proteins from Ebola, HIV, and sialic acidRS-CoV, mediates virus entry through binding to receptor and conformational changes that result in fusion of the viral and target cell membranes
Due to its critical nature, the initial step of influenza entry, HA binding to sialic acid, has received much attention
In this work we characterize the receptor binding properties of the HA from three different subtypes of influenza A: H1, H5 and H9. The H1 HA was from a human source of the recent 2009 outbreak, which represents the first pandemic influenza outbreak of the 21st century. The H5 HA were from a human source, H5 HA/Vietnam (hereafter referred to as H5-V), and an avian source, H5 HA/Qinghai (hereafter referred to as H5-Q), both of which represent highly pathogenic H5 isolates. The H9 HA was from a human source of the 1999 outbreak. We chose to use 2,3-siallylactose (3′SL,
(a) 3′SL chemical structure. (b) 6′SL chemical structure. (c) STD of 3′SL binding to HA from subtypes H1, H5 and H9. (d) STD of 6′SL binding to HA from subtypes H1, H5 and H9. The 1H to be used as reporters are denoted by arrows in (a) and (b). For (c) and (d) the experimental conditions were 2 µM HA, 3 mM 3′SL or 3 mM 6′SL in PBS, pH 7.4 at 25°C with an identical number of scans.
In the STD NMR experiment, the relative intensity of 1H within a particular ligand gives insight into their proximity to the protein interaction surface
Interaction | H3eq | H3ax | Acetyl |
H1∶3′SL |
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0.2 | 1.0 | 0.5 |
H9∶3′SL | 0.3 | 0.3 | 1.0 |
H9∶6′SL | 0.2 | 0.3 | 1.0 |
The H3eq, H3ax and Acetyl resonances of 3′SL occur at 2.73, 1.77 and 2.00 ppm, respectively. The H3eq, H3ax and Acetyl resonances of 6′SL occur at 2.67, 1.70 and 2.00 ppm, respectively. The relative ratio of STD intensity is defined as %STD/%STDmax for a particular HA interaction, where %STDmax corresponds to the largest observed %STD.
In the next step, we performed a STD-based competition assay to determine receptor preference
Experimental conditions were 2 µM HA, 3 mM 3′SL, and 3 mM 6′SL in PBS, pH 7.4 at 25°C with an identical number of scans.
Interestingly, the HA from all three subtypes examined, including those from the highly pathogenic H5, bind to both 3′SL and 6′SL as assayed by NMR. As noted above, HA evolves to utilize receptors found in the target organism (i.e. 3′SL in the avian digestive tract and 6′SL in the human upper respiratory tract,
The STD competition experiments shown in
Finally, it is of interest to consider the structural determinants for sialic acid specificity. In
The numbering corresponds to that of H1 A/California/04/09 HA1. Conserved residues that form interactions with sialic acid
It is next of interest to consider structural differences between H1 and H5-V, for which high-resolution structural data exists
(a) Surface topology of the sialic acid binding site on H1 HA
In the present study we have used STD NMR to establish the relative affinity of HA from three different subtypes for receptor analogs. We found three phenotypes: H1 HA with preference for α2,6-linked sialic acid, H5-V with preference for α2,3-linked sialic acid, and H5-Q and H9 with similar affinity for both sialic acid linkages. Importantly, we also found that each subtype binds to both receptors, which may allow influenza strains to gain a foothold in species with a population of different receptors. Therefore, not only receptor preference but also the overall receptor binding profile of a particular HA subtype may give valuable insight into host tropism, as well as other factors such as fucosylation, sulfation, sialylation and host factor interactions
The HA samples from subtypes H1 A/California/04/09 (H1N1), H5 A/bar-headed goose/Qinghai/1A/05 (H5N1), H5 A/Vietnam/1205/04 (H5N1), and H9 A/Hong Kong/1073/99 (H9N2) were obtained from BEI Resources (Manassas, VA). In each case the HA are full length, prepared in cell culture, and glycosylated. The purity of each HA was verified by SDS-PAGE. The sialic acid receptor analogs, 2,3-sialyllactose (3′SL) and 2,6-sialyllactose (6′SL), were obtained from V-Labs (Covington, LA). For the STD experiments, the experimental conditions were 3 mM sialic acid and 2 µM (monomer) of the respective HA in PBS buffer (pH 7.4). STD NMR experiments were performed with a train of 50 msec gaussian-shaped saturating pulses at 100 Hz power for 3 sec with “on” resonance saturation at −0.4 ppm and “off” resonance saturation at 30 ppm (the relaxation delay was 2 sec before the saturating pulses). The number of scans was 1728 and the spectral width was 14,367 Hz. Spectra were recorded at 25°C on a Bruker AVANCE 900 MHz spectrometer equipped with a cryogenic probe. Spectra were processed by NMRPipe with a 5 Hz line broadening and analyzed by NMRDraw
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