Thermal denaturation of influenza virus and its relationship to membrane fusion.
The X-31 strain of influenza virus was studied by differential scanning calorimetry (DSC), CD and SDS/PAGE analysis as a function of both temperature and pH. A bromelain-treated virus was also studied by these methods. The major transition observed in the intact virus was a result of the denaturation of the haemagglutinin (HA) protein. At pH 7.4, this transition was similar in the intact virus and the isolated HA, but was absent in the bromelain-treated virus. However, at pH 5 the denaturation temperature and enthalpy were both higher for HA in the virus than in the isolated protein, indicating that HA interacts with other molecular components in the intact virus. The transition observed by DSC occurs at a higher temperature than does the thermal transition observed by CD. The temperature of the CD transition coincides with the temperature at which the fusogenicity of the virus increases, and probably corresponds to the formation of an extended coiled-coil conformation. Analysis by SDS/PAGE at neutral pH under non-reducing conditions demonstrates a selective loss of the HA protein trimer, resulting in the formation of aggregates in the range of temperatures of 55 to 70 degrees C. In contrast, at acidic pH, the HA protein is largely in the monomeric form at 25 degrees C, and there is little change with temperature. There is thus a weakening of the quaternary structure of HA at acidic pH prior to heating. At the temperature at which the virus exhibits an increased fusogenicity at neutral pH, there is a loss of secondary structure and a beginning of a destabilization of the trimeric form of HA. This temperature is lower than that required for the major endothermic peak observed in DSC experiments. The results demonstrate that there is no kinetically trapped high-energy form of HA at neutral pH.
Epand
Richard M
RM
Department of Biochemistry, McMaster Health Sciences Centre, Hamilton, ON L8N 3Z5, Canada. epand@mcmaster.ca
Epand
Raquel F
RF
D001963:Bromelains / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion / Q000502:physiology*; D009975:Orthomyxoviridae / Q000737:chemistry* / Q000502:physiology*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D019267:Hemagglutinin Glycoproteins, Influenza Virus; D001963:Bromelains