Characterization of Influenza Hemagglutinin Interactions with Receptor by NMR

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

Characterization of Influenza Hemagglutinin Interactions with Receptor by NMR

<div><p>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.</p> </div

Amino acid sequence alignment for the HA1 of subtypes used in the present work.

<p>The numbering corresponds to that of H1 A/California/04/09 HA1. Conserved residues that form interactions with sialic acid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033958#pone.0033958-Stevens2" target="_blank">[30]</a> are colored green. Non-conserved residues between H1 and H5-V HA are colored yellow. Non-conserved residues between H5-V and H5-Q are shaded red.</p

Relative ratio of STD intensity for HA interactions with 3′SL and 6′SL¹.

1<p>The H_3eq, H_3ax and Acetyl resonances of 3′SL occur at 2.73, 1.77 and 2.00 ppm, respectively. The H_3eq, H_3ax 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/%STD_max for a particular HA interaction, where %STD_max corresponds to the largest observed %STD.</p

Structural determinants of HA receptor preference.

<p>(a) Surface topology of the sialic acid binding site on H1 HA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033958#pone.0033958-Guo1" target="_blank">[33]</a> (PDB entry 3AL4). The side chains of non-conserved residues between H1 and H5-V HA are colored yellow. (b) Surface topology of the sialic aicd binding site on H5-V HA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033958#pone.0033958-Nobusawa1" target="_blank">[31]</a> (PDB entry 2FK0). The side chains of non-conserved residues between H5-V and H5-Q are colored red. In both (a) and (b) the side chains of residues that form interactions with sialic acid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033958#pone.0033958-Stevens2" target="_blank">[30]</a> are colored green. The structures shown for H1 and H5-V were determined in the absence of receptor analog (there are no available structures for HA-receptor complexes of these particular HA; however, there are structures for the HA-receptor complexes of other HA strains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033958#pone.0033958-Skehel1" target="_blank">[7]</a>).</p

Competition STD experiments for 3′SL and 6′SL binding to HA from subtypes H1, H5 and H9.

<p>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.</p

Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding-3

soluble CD81-GST fusion protein. Binding to CD81 was determined by Western Blot analysis of E2 and the GST tag. As a negative control, GST protein without soluble CD81 was incubated with HCV wt. Image is a composite. (B) 293T cells transfected with HCV E1E2 wt or specific mutant expression vectors were lysed 24 h post-transfection. Cleared cell lysate was incubated with AR3A (C1) conformational antibody to assess conformation of mutations. Immunoprecipitated proteins were detected by subsequent Western Blot analysis of E2. Image is a composite.<p><b>Copyright information:</b></p><p>Taken from "Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding"</p><p>http://www.virologyj.com/content/5/1/46</p><p>Virology Journal 2008;5():46-46.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2277408.</p><p></p

Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding-1

Ransfection and used to infect susceptible cell lines (A) Huh7 and (B) Hep3B. Infectivity was measured 72 h pi using a luciferase reporter assay. Infectivity of each mutant is expressed as a percentage of the infectivity observed for the wild-type (wt) H77 HCV E1E2. Values shown are the mean and standard error for a minimum of three assays.<p><b>Copyright information:</b></p><p>Taken from "Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding"</p><p>http://www.virologyj.com/content/5/1/46</p><p>Virology Journal 2008;5():46-46.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2277408.</p><p></p

Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding-2

(B) Incorporation of HCV glycoproteins into HCVpp was determined by pelleting the virus through a 20% sucrose cushion followed by Western Blot analysis. HCV glycoproteins were identified with (α)-E2 and (α)-E1 antibodies. Detection of the HIV p24 capsid protein with an anti-HIV p24 antibody was performed as a loading control. Image is a composite.<p><b>Copyright information:</b></p><p>Taken from "Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding"</p><p>http://www.virologyj.com/content/5/1/46</p><p>Virology Journal 2008;5():46-46.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2277408.</p><p></p

Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding-5

are numbered relative to the AUG start codon of the H77 strain shown and used in this study. The hyperconserved (black rectangles) targeted (asterisk) residues for alanine substitution are indicated.<p><b>Copyright information:</b></p><p>Taken from "Dissecting the role of putative CD81 binding regions of E2 in mediating HCV entry: Putative CD81 binding region 1 is not involved in CD81 binding"</p><p>http://www.virologyj.com/content/5/1/46</p><p>Virology Journal 2008;5():46-46.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2277408.</p><p></p