Abstract
Fusion of influenza viruses with target membranes is induced by acid and involves complex changes in the viral fusion protein hemagglutinin (HA) and in the contact sites between viruses and target membranes (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). At 0 degrees C, in a first, kinetically distinct step, target membranes irreversibly adhere to the viruses. Fusion itself starts only after a lag-phase of several minutes (X-31 strain viruses) or after raising the temperature (PR8/34 strain viruses). We now provide evidence that the initial conformational change resulting in virus-target membrane adhesion is restricted to a (minor) subpopulation of the HA molecules. These molecules become susceptible to bromelain digestion, and they could be labeled with the photoactivatable reagent [3H]PTPC/11, a nonexchangeable lipid present in the target lipid bilayer (Harter, C., Bächi, T., Semenza, G., and Brunner, J. (1988) Biochemistry 27, 1856-1864). Only the HA2 subunit was labeled, and analyses of 2-nitro-5-thio-cyanobenzoic acid fragments derived thereof indicate that the HA2 NH2-terminal segment (fusion peptide) inserted into the target membrane bilayer. When the temperature was raised to trigger fusion of PR8/34 viruses, labeling of HA2 increased by a factor of 130. Most (74%) of that label was incorporated into the COOH-terminal membrane anchor region, but there was also a strong increase (about 30-fold) of NH2-terminal fusion peptide labeling. This suggests that fusion is preceded., or accompanied, by further changes in HA which lead to additional extensive lipid insertions of HA2 fusion peptides.
Highlights
Fusion of influenza viruses with target membranes glycoproteins of which hemagglutinin (HA),’ the fusion prois induced by acidand involvescomplex changes in the tein of influenza virus, is the best characterized (for recent viral fusion proteinhemagglutinin (HA) and in the reviews, see Marsh and Helenius, 1989;Stegmann et al, 1989; contact sites between viruses and target membranes Wilschut and Hoekstra, 1990; White, 1990)
Stricted to a subpopulation of the HA mole- The fusion activity ofHA is triggered by an acid-induced cules. These molecules become susceptible to bromelain conformational change which leads to theexposure of the sodigestion, and theycould be labeled with thephotoac- called fusion peptide, a hydrophobic, 22-residue long NH2
We demonstrate that the HA2 NHp-terminalsegment of PR8/34 and most likely of X-31 hemagglutinin inserts into the targemt embrane bilayer prior to fusion
Summary
Were sedimented in a Sorvall centrifuge (SS 34) at 17,000 rpm (15 min). The sediments (in 2 ml of fusion buffer) were transferred into. PR(7/34 Mount Sinai)) and X-31 (a recombinant strain containing and HA2) was stained with Coomassie Brilliant Blue R-250, and the the HAof the A/Aichi/68 virus) were propagated in the allantoic correspondingbands were excised and subjected to scintillation cavity of embryonated eggs and purified as described before To this end, the polyacrylamide gel slices NTCB Cleauage-Solutions (1-2 ml) of HA2 or BHA2 (1-2 nmol of protein from electroelution of Coomassie Blue-stained preparative SDS-polyacrylamide gels) were concentrated by Centricon microconcentrators (Amicon) to approximately 0.2 ml. Polypeptides (HA2 and NTCB fragments derived thereof) separatedby SDS-polyacrylamidegel electrophoresis were electroeluted (elution buffer 25 mM Tris, 0.19 M glycine, 0.05% SDS, pH8.6).The peptide solutions were concentrated using Centricon microconcentrators (Amicon),and aliquots corresponding to 100200 pmol of peptide were spotted directly onto polyvinyldifluoride membranes.
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