A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur at pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformational change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.Viruses such as influenza that contain lipid membranes appear to enter cells during infection by a process involving the fusion of the viral membrane with a cellular membrane. The results of recent investigations have indicated that, for a number of viruses, fusion occurs optimally over narrow ranges of pH, in the case of influenza viruses between pH 5.0 and pH 5.5 (1-4), and it has been proposed that this correlates with the pH at the site of cell entry in intracellular vesicles such as lysosomes (5).Evidence that the hemagglutinin (HA) glycoprotein is involved in influenza virus-mediated fusion includes the observations that post-translational cleavage ofa precursor HA, HAO, to HA1 and HA2 is required for both virus infectivity (6, 7) and in vitro virus-mediated fusion (4,8) and that the hydrophobic amino-terminal sequence of HA2 is analogous to that of the amino terminus ofthe F1 component of Sendai virus fusion glycoprotein (9-11). Furthermore, the findings that the amino-terminal sequence of HA2 consists of 10 uncharged hydrophobic amino acids (9) and is the most highly conserved sequence in the hemagglutinin (12) suggest that the terminal region may be directly involved in the membrane-fusion reaction. Analysis of the three-dimensional structure (12) of bromelain-released HA (BHA), which lacks the carboxyl-terminal hydrophobic region through which the complete HA is associated with the lipid membrane of the virus particle, suggests that a conformational .change may be required before this can occur.In this investigation, a conformational change of BHA from X-31 (H3N2) influenza virus has been observed, which occurs at pH values corresponding to those optimal 'for the in vitro membrane-fusion activity of the virus. CD, sedimentation, electron microscopy, and proteolytic susceptibility studies have been. used to characterize the pH 5.0-induced transition. The experiments lead to the conclusion that, after incubation at low pH, BHA can form hydrophobic associations with other BHA molecules, with lipid vesicles, or with nonionic detergent micelles. These results are discussed with reference to the threedimensional structure of HA and in relation to its possible role in virus-mediated fusion. METHODSVirus and HA Purification. X-31 (H3N2) influenza virus (13) was grown in embryonated hens' e...

Among critically ill patients and their surrogates, a family-support intervention delivered by the interprofessional ICU team did not significantly affect the surrogates' burden of psychological symptoms, but the surrogates' ratings of the quality of communication and the patient- and family-centeredness of care were better and the length of stay in the ICU was shorter with the intervention than with usual care. (Funded by the UPMC Health System and the Greenwall Foundation; PARTNER ClinicalTrials.gov number, NCT01844492 .).

SUMMARYWe have studied the interactions of synthetic peptides corresponding to the sequence of the amino terminus of the HA2 subunit of influenza virus haemagglutinin with artificial lipid membranes. The peptides could fuse cholesterol-free liposomes at neutral as well as acid pH; however, liposomes containing cholesterol could only be fused below pH 6. The fusion process caused leakage of aqueous liposomal contents. Peptides with amino acid substitutions had fusion properties similar to whole haemagglutinin molecules with the corresponding sequence changes. Non-fusogenic peptides still interacted with the membrane but did not cause leakage of liposomal contents. A correlation between the ~-helical content of peptide and its fusogenicity was noted, but this was not absolute. The results reported here support suggestions for a role of the amino terminus of HA2 in virus-endosome fusion. INTRODUCTIONInfluenza virus haemagglutinin (HA) mediates the fusion of viral and endosomal membranes which is required to initiate infection of cells (reviewed by White et al., 1983;Wharton, 1987). At the low pH of the endosome (between 5 and 6) a conformational change occurs in HA, which is a prerequisite for fusion activity. A number of studies have shown that this change involves a decrease in subunit contacts within the HA trimer (Skehel et al., 1982;Daniels et al., 1983bDaniels et al., , 1985Ruigrok et al., 1986) and the exposure of the highly conserved hydrophobic amino terminus of HA2 (Skehel et al., 1982; Daniels et al., 1983Daniels et al., a, 1985Doms et al., 1985). It has been proposed that this hydrophobic region interacts with lipid membranes during the fusion process and support for this proposition comes from the observation that aggregation of bromelainreleased HA (BHA), which is specifically induced at the pH of fusion, involves the HA2 aminoterminal region (Daniels et aL, 1983a; R. W. H. Ruigrok et al., unpublished results). Also, variant HAs with amino acid substitutions in the amino-terminal region of HA2 constructed by site-directed mutagenesis have fusion and membrane association characteristics different to those of wild-type HA (Gething et al., 1986). In a recently published report, Lear & De Grado (1987) have shown that the peptide corresponding to the amino terminus of HA2 B/Lee/40 is capable of fusing liposomes at neutral pH. In addition, Murata et al. (1987) have demonstrated pH-dependent fusion of liposomes by a peptide corresponding to the amino terminus of HA2 of A/PR/8/34 virus. In the study reported here we synthesized a peptide with the sequence of the 23 amino-terminal residues of HA2 of X31 (H3N2) which was capable of interacting with and fusing membranes. Using specifically modified analogues of this region, we also showed that interaction with membranes was not sufficient for fusion, that the fusion characteristics of peptides were affected by the lipid composition of the liposomes, and that peptides with sequences equivalent to the amino-terminal regions of variants had fusion properties simila...

SUMMARYAt the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.

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Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, KM values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (KI) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.

Summary.-Data from the Los Angeles County Cancer Surveillance Program (CSP) from 1972 to 1975 were used to study the descriptive epidemiology of testicular cancer and prostatic cancer. The very high black/white ratio and late age peak of cancer of the prostate contrasted sharply with the very low ratio and early age peak of testicular cancer. However, both sites had higher rates among upper occupational and social class groupings. Available descriptive and analytical research suggests that the etiology of prostatic cancer is most probably related to hormonal influences rather than to a horizontally transmitted agent, while the etiology of testicular cancer is most probably related to endogenous or exogenous hormonal influences in utero or in infancy, or to in utero exposure to other exogenous agents.