Aptamers selected against various kinds of targets have shown remarkable specificity and affinity, similar to those displayed by antibodies to their antigens. To employ aptamers as genotyping reagents for the identification of pathogens and their strains, in vitro selections were carried out to find aptamers that specifically bind and distinguish the closely related human influenza A virus subtype H3N2. The selected aptamer, P30-10-16, binds specifically to the haemagglutinin (HA) region of the target strain A/Panama/2007/1999(H3N2) and failed to recognize other human influenza viruses, including another strain with the same subtype, H3N2. The aptamer displayed over 15-fold-higher affinity to the HA compared with the monoclonal antibody, and efficiently inhibited HA-mediated membrane fusion. These studies delineate the application of aptamers in the genotyping of viruses.

Interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at McConnell, March 30, 1981 ABSTRACT The molecular mechanism of hemolysis and fusion by influenza virus in acidic media was studied. First, the effect of trypsin treatment on the activity of fibroblast-grown influenza virus was studied. The results showed that the split form of viral hemagglutinin, HA1 and HA2, but not the precursor, is responsible for the activity. Second, the interaction of egg-grown influenza virus, which contains the split hemagglutinin, with lipid liposomes was studied by spin labeling and electron microscopy. Phospholipid transfer from the viral envelope to the lipid bilayer membrane occurred within 30 s at pH 4.5-5.4. The transfer is largely independent ofthe lipid composition and the crystalline vs. liquid/ crystalline state ofthe membrane. Virus-induced lysis ofliposomes also took place rapidly in the same pH range. Envelope fusion with liposomes occurred at pH 5.2 but not at pH 7.0. These characteristic interactions were similar to those between influenza virus and erythrocytes reported previously. On the other hand, hemagglutinating virus ofJapan did not interact with liposomes at neutral pH. These results suggest that protonation of the NH2-terminal segment of the HA2 form causes interaction of the segment with the lipid core ofthe target cell membrane, leading to hemolysis and fusion.

A pulse saturation-recovery electron paramagnetic resonance (EPR) method has been developed that allows estimation of the exchange rates of a spin-labeled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a function of concentrations of both molecular oxygen and the spin label. Influenza viral membrane, one of the simplest paradigms for the study of biomembranes, showed the presence of two membrane domains with slow and fast collision rates with oxygen (a 16-fold difference) at 30 degrees C. The outbound rate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 micros residency time), respectively, indicating that the SLOT domain is highly dynamic and that the entire SLOT domain represents about one-third of the membrane area. Because the oxygen transport rate in the SLOT domain is a factor of two smaller than that in purple membrane, where bacteriorhodopsin is aggregated, we propose that the SLOT domain in the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase.

Hemagglutinin (HA) glycoproteins isolated from influenza virus caused hemolysis and liposome lysis at pH < 6.0. The pH dependence was similar to that of the parent virus. Hemagglutination and hemolysis titers of HA were comparable with those of virus. The time course of hemolysis by HA was somewhat different from that by virus. HA did not cause fusion of erythrocytes in acidic media, in contrast to virus. Both HA and virus, previously incubated at pH < 6.0, lost their low-pH-induced hemolytic activity. Isolated HA formed rosette-like structures at neutral pH, and these aggregated in acidic media. Virus also aggregated in acidic media and its envelope became leaky to negative stain. HA previously incubated at pH < 6.0 became susceptible to trypsin digestion. Both reversible and irreversible structural changes of HA were observed by fluorescence spectroscopy; a reversible change at a pH between neutral and 6.4 and an irreversible one at pH < 6.0. Bromelain-released HA did not cause hemolysis and liposome lysis in acidic media. The precursor form of HA did not have hemolytic activity in acidic media. The similarity in pH dependence indicates that the structural change in HA induced at pH < 6.0 is the cause of activation and inactivation of hemolysis, HA and virus aggregation, and trypsin susceptibility. We propose that the hydrophobic NH2-terminal segment of HA2 is exposed during the structural change and interacts with the target membranes, causing a permeability increase and leading to hemolysis and lysis. The virus-induced hemolysis can be ascribed for the most part to envelope fusion activated in acidic media.Low pH-induced hemolytic activity of influenza virus has recently been observed (1-3). Based on this finding, we proposed an infectious cell entry mechanism of the virus (4), which is essentially the same as that proposed and developed for Semliki Forest virus (5)-i.e., uptake by endocytosis and fusion of endocytosed vesicles with lysosomes, followed by fusion of the virus envelope with the secondary lysosomal membrane because of the low pH. Recently, studies supporting this entry mechanism for influenza virus have been reported (6,7). Hemagglutinin (HA) glycoproteins on the virus envelope were suggested as the molecules responsible for the low pH-induced activity (1, 4). In the present study, we focused on the function of HA isolated from virus in comparison with that of the parent virus. We also studied functions of bromelain-released HA (BHA), which lacks the COOH-terminal hydrophobic anchoring peptide (8), and of the precursor form of HA, HAo. MATERIALS AND METHODSInfluenza virus AOPR8 was grown in embryonated chicken eggs and purified as described (9). Virus was also grown in a monolayer culture of chicken embryo fibroblasts to obtain HAo (4). The virus was suspended by Pipes/saline (135 mM NaCI/10 mM Pipes, pH 7.4) and stored at -80°C.HA was isolated by solubilization with detergent and centrifugation in sucrose density gradients. Virus was treated with Triton X-100 (4 mg/mg of viral protein...

Rotational diffusion of erythrocyte anion channel protein band 3 was measured in ghost membranes by observing time-resolved phosphorescence anisotropy decays of eosinyl-5-maleimide covalently attached to the protein. Experiments were carried out under conditions similar to those employed by Tsuji and Ohnishi (1986) for translational diffusion measurement of band 3 [(1986) Biochemistry 25, 6133-6139] to allow direct comparison of rotational and translational diffusion of band 3. Detailed analysis of diffusive properties of band 3 in ghost membranes was made on the basis of these rotational and translational diffusion data. Rotational diffusion measurements indicated that there are at least three populations of band 3 molecules with high, low, and no rotational mobilities in the time scale of 10(-4)-10(-2) s. These populations are in equilibrium, and the fractional ratios are strongly temperature dependent. At 26 degrees C, 44% of band 3 molecules are mobile (16% have an average rotational correlation time of 0.19 ms, and 28% have an average correlation time of 2.4 ms), and 56% are immobile. These results correlate well with translational diffusion data which indicated 40% mobile and 60% immobile fractions of band 3. The rotational diffusion data together with the translational diffusion data by Tsuji and Ohnishi (1986) and Golan and Veatch [(1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2537-2541] suggest that immobilization of band 3 is largely caused by binding of band 3 oligomers to ankyrin, which abolishes both rotational and translational diffusion of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)