Probing the metastable state of influenza hemagglutinin

Abstract

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

Keywords: glycoprotein structure; hemagglutinin; influenza virus; nuclear magnetic resonance (NMR); peptide conformation; virus.

Figure 1.

Discovery of a peptide inhibitor of HA function. a, inhibition of H7 HA-mediated entry by H7 HA peptides at 100 μm. Relative inhibition is plotted as a function of the ratio of VSVG VLP entry to H7 HA-mediated VLP entry. The six most potent peptides are denoted in red; inhibitory peptides discarded due to cytotoxicity are denoted by asterisks. b, amino acid sequences of the six most potent peptides. Numbering corresponds to that of H3 HA (8). c, location of inhibitory peptides within the H7 HA structure (Protein Data Bank code 4R8W). The six most potent peptide sequences are highlighted in red on one monomer of the HA trimer.

Figure 2.

PEP87 inhibits H5 and H7 HA entry. a, dose-dependent inhibition of H5 and H7 HA-mediated entry by PEP87. The relative entry levels are based on pseudovirus entry into HEK 293T cells measured by a luciferase-based assay in the presence of varying amounts of PEP87. The dotted lines correspond to fits of the data to the following equation: Percent entry = 100/(1 + [PEP87]/IC₅₀). b, cytotoxicity of PEP87 as a function of peptide concentration. Cellular cytotoxicity was measured to be proportional to the overall ATP levels present in cells treated with increasing concentrations of PEP87 (0–250 μm). c, sequence alignment of H5 and H7 HA PEP87 regions. The data represented in a and b are the averages of experiments performed in triplicate where the error bars represent ±S.D.

Figure 3.

Mutagenesis studies of PEP87 inhibition. a, relative inhibition of wild-type and select site-directed mutants of HA-mediated entry by PEP87. Numbering corresponds to that of H3 HA (8). The data represented here are averages of experiments performed in triplicate (error bars represent ±S.D.). b, location of mutations in H5 HA (Protein Data Bank entry 2FK0). PEP87 sequence is highlighted in yellow, and the mutation sites are depicted in red and blue. Mutation sites in red are more sensitive to PEP87 inhibition, whereas those in blue are less sensitive.

Figure 4.

Characterization of PEP87 structure by CD. a, ellipticity of PEP87 in the presence and absence of 40% TFE. CD spectra were recorded from 190 to 260 nm at 100 μm (0.2 mg/ml) PEP87 in 20 mm phosphate, pH 8.0, ± 40% TFE at 25 °C. b, ellipticity of PEP87 as a function of peptide concentration in 20 mm phosphate, pH 8.0, at 25 °C.

Figure 5.

NMR characterization of PEP87 in the presence of cosolvent. a, secondary chemical shift of ¹³C_α. Random coil chemical shifts are taken from Merutka et al. (14). b, ¹H-¹H NOESY spectrum for the H_N region. Cross-peaks are denoted by PEP87 residue numbers. c, minimized mean structure of PEP87. d, electrostatic profile of the minimized mean structure of PEP87. Experimental conditions were 200 μm PEP87 in 20 mm phosphate, pH 7.5, 50 mm NaCl, 40% TFE-d, 10% ²H₂O at 25 °C.

Figure 6.

STD NMR binding studies of PEP87 to HA. a, reference ¹H 1D NMR of 200 μm PEP87 in 50 mm phosphate, pH 8.2, 50 mm NaCl in 100% ²H₂O at 25 °C. b, STD NMR of PEP87 binding to recombinant H5 HA. Experimental conditions were 10 μm HA and 200 μm PEP87 in 50 mm phosphate, pH 8.2, 50 mm NaCl in 100% ²H₂O at 25 °C. c, STD NMR competition assay between α2,3-sialyllactose and PEP87 (shown in dashed lines). Experimental conditions were 10 μm HA and 3 mm α2,3-sialyllactose ± 200 μm PEP87 in 50 mm phosphate, pH 8.2, 50 mm NaCl in 100% ²H₂O at 25 °C.

Figure 7.

Disruption of native HA structure upon binding PEP87 as assayed by intrinsic tryptophan fluorescence and chemical cross-linking experiments. a, fluorescence plots of HA, 50 μm PEP87, and HA + 50 μm PEP87 and the resulting plot when the mixture is corrected for PEP87 fluorescence contribution. Sample conditions were ±2.5 μm HA with ±50 μm PEP87 in 50 mm phosphate, pH 8.2, 50 mm NaCl. b, the corrected fluorescence plots of HA fluorescence exhibiting the effects of increasing the PEP87 concentration from 0 to 250 μm. c, SDS-PAGE of recombinant H5 HA in the absence and presence of chemical cross-linkers and PEP87. Lane 1 corresponds to the molecular weight markers.