title: Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin.
abstract: The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.
authors.affiliation: Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Room 2339, Bethesda, Maryland 20892-8012, USA .
authors.forename: David P
authors.initials: DP
authors.lastname: Remeta
authors.forename: Mathias
authors.initials: M
authors.lastname: Krumbiegel
authors.forename: Conceição A S A
authors.initials: CA
authors.lastname: Minetti
authors.forename: Anu
authors.initials: A
authors.lastname: Puri
authors.forename: Ann
authors.initials: A
authors.lastname: Ginsburg
authors.forename: Robert
authors.initials: R
authors.lastname: Blumenthal
mesh_terms: D000143:Acids; D002942:Circular Dichroism; D004355:Drug Stability; D004910:Erythrocyte Membrane; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008561:Membrane Fusion / Q000502:physiology; D009975:Orthomyxoviridae / Q000472:pathogenicity / Q000502:physiology; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000143:Acids; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D014364:Tryptophan