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. 1999 Jun;73(6):4567-74.
doi: 10.1128/JVI.73.6.4567-4574.1999.

Conformational intermediates and fusion activity of influenza virus hemagglutinin

T Korte  1 , K Ludwig, F P Booy, R Blumenthal, A Herrmann
Affiliations

Conformational intermediates and fusion activity of influenza virus hemagglutinin

T Korte et al. J Virol. 1999 Jun.

Abstract

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824-3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37 degrees C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20 degrees C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37-43, 1994).

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Figures

FIG. 1
FIG. 1
Influence of low-pH preincubation on the rate constant of influenza virus-ghost fusion at pH 5.0 and 37°C in sodium acetate buffer. (A) influenza virus X31; (B) A/Japan/305/57; (C) A/PR 8/34. Preincubation of R18-labeled influenza virus was done at low pH (●, pH 5.0; ■, pH 5.2; ▾, pH 5.4) and 37°C in the absence of target membranes for the indicated times. Subsequently, the virus sample was reneutralized (pH 7.4) and binding to ghost membranes was performed on ice for 30 min as described in the text. After binding, the fusion was measured at pH 5.0 and 37°C by fluorescence dequenching of R18. To compare the results between the various strains, we have normalized the rate constants to that of the control (set at 100%), which corresponds to the fusion at pH 5.0 and 37°C without any preincubation of viruses at low pH. Fluorescence was measured at excitation and emission wavelengths of 560 and 590 nm, respectively (time resolution, 0.5 s). Note the different scaling of the preincubation times.
FIG. 2
FIG. 2
Influence of preincubation of influenza virus X31 (■) and A/Japan/305/57 (▾) at pH 5.0 and 20°C on the rate constant (A) and extent (B) of virus-ghost fusion at pH 5.0 and 37°C in sodium acetate buffer. Preincubation of influenza virus was done at pH 5.0 and 20°C in the absence of target membranes for the indicated times. Subsequently, the virus sample was reneutralized (pH 7.4) and binding to ghost membranes was performed on ice as described in the text. After binding, the fusion was measured at pH 5.0 and 37°C. To compare the results between the various strains, we have normalized the data to the control (set at 100%), which corresponds to the fusion at pH 5.0 and 37°C without any preincubation of viruses at low pH.
FIG. 3
FIG. 3
Influence of temperature on the low-pH inactivation of influenza virus A/PR 8/34 and on the rate constant (A) and extent (B) of virus-ghost fusion at pH 5.0 and 37°C in sodium acetate buffer. Preincubation of influenza virus was done at pH 5.0 and 3°C (▾), 20°C (■), and 37°C (●) in the absence of target membranes for the indicated times. Subsequently, the virus sample was reneutralized (pH 7.4) and binding to ghost membranes was performed on ice as described in the text. After binding, the fusion was measured at pH 5.0 and 37°C. To compare the results between the various strains, we have normalized the data to the control (set at 100%), which corresponds to the fusion at pH 5.0 and 37°C without any preincubation of viruses at low pH.
FIG. 4
FIG. 4
pH dependence of the bis-ANS fluorescence in the presence of isolated HA and of the extent of virus-ghost fusion of influenza viruses of different subtypes (37°C). (A) Kinetics of the bis-ANS fluorescence in the presence of HA of A/PR 8/34 (data not shown for X31 and A/Japan/305/57). At time zero, HA (4 μg/ml) was added to buffer (37°C) containing 3.25 nmol of bis-ANS per ml. Fluorescence was measured at excitation and emission wavelengths of 400 and 490 nm, respectively (time resolution, 0.5 s). The relative fluorescence, Irel, is shown (see Materials and Methods). (B) Maximum of the relative fluorescence, Irel, of bis-ANS after 10 min of incubation at the desired pH. (C) Virus-ghost fusion. Fusion was measured at the indicated pH and 37°C for 20 min by the FDQ assay with the lipid-like fluorophore R18 (see the legend to Fig. 1). ▾, A/Japan/305/57; ■, X31; ●, A/PR 8/34. (Inset to panel B) Comparison of bis-ANS fluorescence in the presence of isolated HA of A/PR 8/34 (●) or intact A/PR 8/34 (○). Bis-ANS fluorescence in the presence of intact viruses (20 μg of virus protein/ml) was measured as described for isolated HA.
FIG. 5
FIG. 5
Temperature dependence of the bis-ANS fluorescence, Inorm, at pH 5.0 in the presence of HA (4 μg/ml) of different influenza virus subtypes. ▾, A/Japan/305/57; ■, X31; ●, A/PR 8/34. The maximum of the relative fluorescence, Irel, of bis-ANS (3.25 nmol/ml) after 10 min of incubation at pH 5.0 at the desired temperature is shown. For details, see the legend to Fig. 4 and Materials and Methods.
FIG. 6
FIG. 6
Reversibility of the low-pH fluorescence of bis-ANS in the presence of HA of A/Japan/305/57 (4 μg/ml). After preincubation of HA at pH 5.0 (▾), pH 5.2 (▴), or pH 5.4 (■), the fluorescence was measured at pH 7.4 for 15 min. The relative fluorescence, Irel, is presented (see equation 1 in Materials and Methods; I(t) corresponds to the fluorescence intensity after neutralization). All steps were done at 37°C. The concentration of bis-ANS was 3.25 nmol/ml. For details, see the legend to Fig. 4 and Materials and Methods.
FIG. 7
FIG. 7
Cryoelectron micrographs of whole influenza virus X31 (2 mg/ml) after incubation at 37°C and pH 7.4 (A), pH 5.3 (B), and pH 4.9 (C).
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