The N terminus of H3-influenza hemagglutinin as a site-of-vulnerability to neutralizing antibody.

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. https://doi.org/10.7554/eLife.00333.001 eLife digest Influenza is caused by viruses that infect birds and mammals. These viruses enter cells when two lipid bilayers—one surrounding the virus, the other enclosing the cellular compartment into which the virus has been engulfed—merge to form a single unified membrane. This process, known as membrane fusion, allows the RNA of the virus to gain access to the host cell's molecular machinery, which it commandeers to produce multiple copies of itself and to direct the assembly of new virus particles. The process of membrane fusion generally includes an intermediate hemifused state in which only the adjacent monolayers from each bilayer have merged. In addition to its role in virology, membrane fusion is critical for many other biological processes, including exocytosis, protein trafficking and the fertilization of eggs by sperm. Efficient membrane fusion requires a catalyst, and a glycoprotein known as the influenza hemagglutinin performs this role for the influenza virus. The hemagglutinin is found on the surface of the virus, and a typical influenza virus particle can have a few hundred such molecules on its surface. When an influenza virus particle binds to the surface of a cell (as a result of these hemagglutinin molecules interacting with cellular receptor molecules), the cell engulfs the virus into an internal compartment called an endosome. Acidification of the endosome, part of the cell's normal activity, triggers a sequence of conformational changes in the hemagglutinin molecules on the surface of the virus. One part of the hemagglutinin inserts itself into the endosomal membrane, and further conformational changes draw the endosomal and viral membranes together into an intermediate, hemifused state; the process then continues until fusion of the two membranes is complete. Previous work has suggested that an average of three hemagglutinin molecules are required to fuse the endosomal and viral membranes. Ivanovic et al. have now investigated the molecular details of this process and described the time course of conformational changes undergone by the hemagglutinin molecules from the moment the pH is lowered within the endosome until the time when hemifusion of the endosomal and viral membranes is complete. They find, among other things, that hemifusion proceeds rapidly only when three or four immediately adjacent hemagglutinin molecules have inserted into the endosomal membrane. Since membrane fusion is a very general cellular process, the findings of Ivanovic et al. are relevant to many areas of cell biology, in addition to having potential applications in virology. https://doi.org/10.7554/eLife.00333.002 Introduction Fusion of two lipid-bilayer membranes is a thermodynamically favorable process, but it crosses a high kinetic barrier as the two bilayers approach each other. Efficient fusion therefore requires a catalyst, a role served in living cells by a fusion protein or protein complex. The influenza virus hemagglutinin (HA) has become an important paradigm of a fusion catalyst, in part because of early crystallographic and mechanistic studies and in part because of continued concern about a virus that caused tens of millions of deaths during the 20th century. HA facilitates fusion by undergoing a large-scale conformational change, coupled to the two fusing membranes (virus and target). Our current picture of HA-mediated membrane fusion, illustrated in Figure 1A (Harrison, 2008), comes from HA structures in both pre- and post-fusion conformations and from inferences about transient intermediate states. HA is a homotrimer, synthesized as an inactive precursor, [HA0]3, and activated for fusion by proteolytic cleavage of each chain into HA1 and HA2, yielding [HA1-HA2]3. At the N-terminus of HA2 is a hydrophobic ‘fusion peptide', which following cleavage inserts firmly into a pocket near the axis of the trimer (Chen et al., 1998). Exposure to low pH, which during infection occurs in an endosome, causes the HA1 ‘head' to separate from the HA2 ‘stem' and enables a set of HA2 conformational transformations: (1) release of the fusion peptide from its pre-fusion pocket; (2) HA2 extension; (3) insertion of the fusion peptide into the target membrane; (4) fold-back of the extended HA2 intermediate (Figure 1A) (Skehel and Wiley, 2000). This last step brings together the fusion peptide and the C-terminal, transmembrane segment of each HA2, anchored respectively in the viral and target membranes, which thus approach each other, either as apposed protrusions or as a single, target-membrane protrusion (Kuzmin et al., 2001; Lee, 2010). Fusion then ensues, initially as hemifusion (merger of only the proximal membrane leaflets) and then as formation of a continuous aqueous channel. Figure 1 with 1 supplement see all Download asset Open asset Single-virion analysis of fusion-promoting conformational change in influenza virus HA. (A) Hydrophobic fusion peptide (red asterisk) is initially inserted into a pocket near the trimer threefold axis. HA assumes an ‘open' conformation upon proton binding allowing fusion-peptide release and membrane insertion. The fold-back of the extended intermediate causes hemifusion. The known pre-fusion and post-fusion HA structures are colored, and the inferred structural transitions are showed in gray. (B) Left: Tile view from pH drop (t0) of a virion initially displaying directed motion (white arrow) followed by arrest (ta) then hemifusion (th). Red circle marks the final virion location. Arrowheads mark two virions that were arrested at pH drop and hemifused at or just before t40 s and t68 s respectively. Right: Fluorescence trace of the virion circled in (A) (red line), line fitting the timing of virion arrest at its target location (black line) and parameters t0 (green vertical line), ta (blue horizontal line) and th (orange horizontal line) and arrest to hemifusion, ta–h (dark orange horizontal line). (C) tlag(pHdrop–arrest) for all virions for which arrest values could be derived (left) and tlag(arrest–hemifusion) for all virions for which both arrest and hemifusion values could be derived (right) with gamma-distribution fit (black line). Data are pooled from three independent experiments. (D) Mean tlag(pHdrop–arrest) and tlag(arrest–hemifusion). Error bars represent the standard error of the mean. (E) and (F) N derived from fitting tlag(pHdrop-arrest) (E) and tlag(arrest–hemifusion) (F) with gamma probability density. (G) Rate constants derived from fitting tlag(arrest–hemifusion) and tlag(pHdrop–arrest) with gamma probability density and keeping N fixed (N = 3) for both processes. (B–G) X31-HA virions have X31 HA in otherwise Udorn genetic background. Data shown are from representative experiments performed on the same day (n = 50 to 150) unless indicated that multiple experiments were pooled. Error bars represent 95% confidence interval for gamma fit-derived values unless otherwise indicated. Please refer to Figure 1—figure supplement 1 for all histogram and gamma-distribution fit data plotted in (D–G). https://doi.org/10.7554/eLife.00333.003 We report here a series of experiments that probe the relationship between HA structural properties and kinetic intermediates in the fusion pathway. Floyd et al. (2008) devised a method to monitor in real time the fusion of individual influenza virus particles with planar bilayers. Their results led to the conclusion that fusion requires on average three HA trimers, each of which independently undergoes the same, rate-limiting rearrangement, but they left undetermined the relationship between this conclusion and the inferred intermediates in Figure 1A. In the experiments reported here, we correlate HA structure with observed variations in fusion kinetics by comparing rates for appropriate HA mutants. We conclude that irreversible engagement of fusion peptides from 3–4 neighboring [HA1-HA2]3 trimers, within a much larger virus-target-membrane interface, leads to subsequent rearrangements that rapidly and cooperatively induce membrane merger. Release of the fusion peptide from its pocket is rate-limiting for membrane engagement. A long-lived membrane-inserted extended intermediate is a fundamental aspect of the fusion mechanism. Results Hemifusion times and particle arrest We recorded in real time a large number of individual influenza virions (approximately 1000 virions per field of view), labeled with a lipophilic fluorophore (R18), as they fused with a supported planar bilayer (see ‘Materials and methods'). We followed hemifusion as a spike in individual virion fluorescence resulting from R18 fluorescence dequenching upon dilution into the target membrane (Figure 1B). We also observed a previously undetected fusion intermediate. Upon initiating the flow of buffer for pH exchange, most virions started to move under the hydrodynamic force while retaining contacts with the target bilayer. The particles arrested at various times after the pH drop, but invariably preceding hemifusion (Figure 1B; Video 1). The arrest was irreversible (Videos 2 and 3). A subset (approximately 20% at pH 5.5 and 5.65) of virions had already arrested at the onset of imaging or had done so before the pH drop. For lower final pH, a larger fraction had arrested by the time the pH drop was complete (Table 1). We determined lag times between pH drop and individual virion arrest (tlag(pHdrop–arrest)) and between arrest and hemifusion (tlag(arrest–hemifusion)) whenever both values could be extracted from the data (Figure 1B,C), for a range of proton concentrations between 2 and 100 µM (pH 5.65–4) (Figure 1D). Mean values for tlag(pHdrop–arrest) and tlag(arrest–hemifusion) show the same pH dependence, with mean tlag(pHdrop–arrest) being about an order of magnitude shorter throughout the tested pH range. Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg X31-HA WT virion hemifusion at pH 5.5 from t0 to t230 s at 20× the actual rate. https://doi.org/10.7554/eLife.00333.005 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Virion arrest is an irreversible intermediate of fusion. X31-HA WT virions were imaged. pH was dropped from 7.4 to 5.65 then brought back up to 7.4. Buffer flow was kept constant except when it was stopped to allow for the source buffer exchange back to neutral (between approximately t45 s and t95 s). https://doi.org/10.7554/eLife.00333.006 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Virion arrest is an irreversible intermediate of fusion. A different field of view of the same experimental lane used in Video 2 after the events imaged in Video 2. pH was dropped to pH 5.65. There is a marked reduction in the mobile fraction in Video 3 relative to early times shown in Video 2. Furthermore, prearrested virions proceeded to hemifusion despite the intermediate reneutralization step. The videos are shown at 20× the actual rate. https://doi.org/10.7554/eLife.00333.007 Table 1 Arrest and hemifusion statistics for X31-HA WT and arrest statistics for S4GHA2Udorn virions https://doi.org/10.7554/eLife.00333.008 Virion arrest to hemifusion*pH drop to virion arrest†pH 5.5X31-HA WT‡X31-HA WT‡S4GHA2Udorn§Number of virions3801215401Mean lag time (s)#105.1 ± 2.710.3 ± 0.29 ± 0.4N¶3.7 ± 0.63.4 ± 0.23.4 ± 0.3Rate constant (s−1) ¶0.032 ± 0.0060.33 ± 0.020.37 ± 0.03Mobile at pH drop**81%76%Mobile that hemifused**75%65%Static that hemifused**82%81%pH 4.5Number of virions155392385Mean lag time (s) #13.3 ± 0.61.4 ± 0.0041.4 ± 0.004Mobile at pH drop**46%38% * tlag(arrest–hemifusion) for all virions for which both arrest and hemifusion values could be derived. † tlag(pHdrop–arrest) for all mobile virions for which arrest values could be derived. ‡ X31-HA WT data are pooled from three independent experiments. § S4GHA2Udorn data are pooled from two independent experiments. # Errors represent the standard error of the mean. ¶ Errors represent the 95% confidence interval for the values derived from gamma-probability fits shown in Figure 1C (X31-HA virions) or Figure 4C (S4GHA2Udorn virions). ** Percentages are derived from entire data sets. We can fit the distributions for both tlag(pHdrop–arrest) and tlag(arrest–hemifusion) with one describing a requirement for N independent events (either parallel or sequential; Feller, 1968), each with rate constant, k (Figure 1C and Table 1). For any given particle, the two lag times are uncorrelated, ruling out any mutual dependence on overall particle properties such as length (Figure 2). We have further controlled for virion length by using only shorter-virion fractions in our experiments (see ‘Materials and methods'). From the distributions, we derive N = 3–4 both for the number of events required to arrest virions following pH drop (Figure 1E and Table 1) and for the number of events required for hemifusion of the arrested particle (Figure 1F and Table 1) (see ‘Materials and methods'); the latter is similar to the value obtained previously from tlag(pHdrop–hemifusion) distributions (Floyd et al., 2008). Figure 2 Download asset Open asset Virion arrest and hemifusion lag times are uncorrelated. tlag(arrest–hemifusion) vs tlag(pHdrop–arrest) for X31-HA virions at pH 5.5 (n = 380). Data are pooled from three independent experiments also shown in Figure 1C, Table 1 and Figure 4A,B (X31–HA). https://doi.org/10.7554/eLife.00333.009 A simple interpretation of virion arrest is that insertion of fusion peptides from a number of independent HA trimers (3 or 4) into the target membrane anchors the particle. Bulk experiments have shown that short incubations of virions with target membranes at pH 5 and 0°C lead to insertion of a small subset of fusion peptides and stable virus anchoring not associated with membrane fusion (Tsurudome et al., 1992). We attribute the majority of pre-arrested events during high pH experiments (pH 5.5 and above) to imperfections in the bilayer or defective virions; the observed increase in the immobile fraction at lower pH might result from genuine triggering and membrane insertion of HA fusion peptides upon proton binding during the pH transition (see ‘Materials and methods'). Site-directed HA mutations and the rate-limiting step for HA rearrangement To probe the molecular mechanism of arrest and its relationship to the mechanism of hemifusion (for which membrane insertion of the fusion peptide is clearly critical), we generated recombinant virus particles with site-directed mutations in HA. We used a set of plasmids derived from the A/Udorn/72 H3 influenza strain. The HA of Udorn is 97% identical in amino-acid sequence to that of X-31, the virus used in previous experiments (Floyd et al., 2008) and also the source of HA in otherwise Udorn genetic background in the experiments in Figure 1. Nonetheless, recombinant virions with Udorn HA had shorter hemifusion lag times in the physiologically relevant pH regime (pH > 4.5) than did those with X-31 HA (Figure 3A, Videos 1 and 4). Moreover, the Udorn-HA particles did not show directed motion under flow at any observable time point—that is, they had arrested by the time the low-pH transition was complete (see ‘Materials and methods'). The Udorn hemifusion lag time (tlag(pHdrop–hemifusion)) distribution between pH 5.65 and 4 gave a pH-independent value of N close to 3 (Figure 3B), showing that differences between Udorn and X31 HAs do not affect the number of rate-limiting steps between virion arrest and hemifusion, but only their individual rate constants (Figure 3C). Figure 3 with 1 supplement see all Download asset Open asset Udorn virions have accelerated hemifusion kinetics. (A) Mean tlag(pHdrop–hemifusion) for Udorn and mean tlag(arrest–hemifusion) for X31-HA virions. Error bars represent the standard error of the mean. (B) N derived from fitting tlag(pHdrop–hemifusion) with gamma probability density. (C) Rate constants derived from fitting tlag(pHdrop-arrest) for Udorn and tlag(arrest–hemifusion) for X31-HA virions with gamma probability density and keeping each N fixed (N = 3). (B–C) Error bars represent 95% confidence interval for gamma fit-derived values. (A–C) Data shown are from representative experiments performed on the same day (n = 50 to 350). Please refer to Figure 3—figure supplement 1 for all histogram and gamma-distribution fit data plotted in (A–C). (D) Top left: Ribbon representation of X31-HA trimer (Weis et al., 1990) showing positions of all residues that differ in Udorn-HA (arrowhead) including Gly4HA2 (asterisk). Top right: Cartoon of an HA monomer emphasizing Asp112HA2-fusion peptide hydrogen bond network and showing positions of residues along the HA2 chain. Bottom: Close-up of the Asp112HA2-fusion peptide network (region marked with an orange square on top left). https://doi.org/10.7554/eLife.00333.010 Comparison of the Udorn and X-31 HA amino-acid sequences suggests that one particular difference might account for the accelerated fusion kinetics of the former—a substitution of serine for glycine at position four in HA2. Udorn is the only strain in the database with this substitution, glycine being otherwise universally conserved (Nobusawa et al., 1991; Cross et al., 2009). In the pre-fusion conformation of HA, Gly4HA2 participates in a network of polar hydrogen-bond interactions with the carboxylate of Asp112 HA2, which is also strictly conserved (Figure 3D) (Wilson et al., 1981; Weis et al., 1990; Russell et al., 2004). A serine substitution would weaken or interrupt this interaction, because the glycine has a backbone conformation not allowed for other residues. Previous studies have reported an elevated pH threshold of fusion for cell-surface expressed HA at or Asp112 et al., et al., Gly4HA2 might also a conformation of the membrane-inserted fusion-peptide et al., but its does not critical for fusion-peptide insertion into membranes et al., We generated a series of HA in the of recombinant to the for fusion kinetics of the and of the fusion-peptide in activated HA. These and as as in both Figure 4A,B and Videos 1 and our analysis of hemifusion rates for these at pH The hemifusion, and the S4GHA2Udorn it (Figure Moreover, WT Udorn virions do not show motion at pH drop, while S4GHA2Udorn virions do (Videos 5 and Table 1). parameters for derived from the tlag(pHdrop–arrest) distribution for the S4GHA2Udorn particles are from those for X31-HA WT (Figure 4C and Table 1). These show that a single and thus the same molecular process, both the rate of arrest and the rate of hemifusion. Figure 4 with 1 supplement see all Download asset Open asset release from its pre-fusion pocket is a rate-limiting molecular rearrangement in the physiologically relevant pH (A) pH mean tlag(arrest–hemifusion) for X31-HA S4GHA2Udorn and mean tlag(pHdrop–hemifusion) for Udorn and (B) N derived from fits of the data also in (C) of tlag(arrest–hemifusion) distribution for S4GHA2Udorn virions at pH 5.5 with the fit (black line). (D) pH mean tlag(pHdrop–hemifusion) for indicated virions. (E) N derived from fits of the data also in Data shown are from pooled independent experiments for Udorn and X31-HA WT and for Udorn and X31-HA virions; = to Error bars represent the standard error of the mean and or the 95% confidence interval for gamma fit-derived values and Please refer to Figure supplement 1 for all histogram and gamma-distribution fit data plotted in and Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Udorn WT virion hemifusion at pH 5.5 from t0 to s at 20× the actual rate. Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg virion hemifusion at pH 5.5 from t0 to s at 20× the actual rate. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg S4GHA2Udorn virion hemifusion at pH 5.5 from t0 to s at 20× the actual rate. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg virion hemifusion at pH 5.5 from t0 to t40 s at 20× the actual rate. Video Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg virion hemifusion at pH 5.5 from t0 to s at 20× the actual rate. Data from that the has an important on hemifusion rate at pH mutations hemifusion of both Udorn and X31-HA but the on the latter is (Figure We from these that the contacts between the fusion-peptide residues and and that the change then this source of of the mutations change the values of N derived from the pH 5.5 tlag(arrest–hemifusion) distributions for tlag(pHdrop–hemifusion) (Figure showing that these changes do not affect the number of We conclude that for these release of the fusion peptide from its pre-fusion pocket is a rate-limiting step in the transition from arrest to hemifusion. The changes in fusion kinetics by the including the fit obtained from a probability distribution that assumes changes in the rates of all N events (see ‘Materials and further support our that N is to the number of HA trimers that independently their fusion peptides before a transition can The rate of arrest for either of the was for to detect motion (Videos and We therefore could not N from arrest distributions for those but we could that fusion-peptide release from its pocket is rate-limiting for virion arrest as as for hemifusion. At pH and the described have mean values for tlag(pHdrop–hemifusion) (Figure Moreover, the lag times have become pH because most of the relevant have become A subset than at pH of X31-HA WT and S4GHA2Udorn virions directed motion under the force of buffer flow at the pH drop, both with mean times of just under s (Table 1). the tlag(pHdrop–hemifusion) distributions with a gamma probability density values of N (between 3 and for all the that changes with pH is not the number of rate-limiting rearrangements to hemifusion but only their rate (Figure of Asp112 mutations that affect of the fusion peptide in its pre-fusion pocket and the fusion rate at pH 5.5 have or on the fusion rate at pH and The value of N and the that a fraction of the X31-HA WT and S4GHA2Udorn particles show directed motion after the pH drop that at pH rate-limiting rearrangement membrane insertion of the fusion The of mutations at positions 4 and in HA2 on hemifusion at the physiologically relevant pH of 5.5 that the steps the rate of arrest the time of pH and the rate of transition from arrest to hemifusion are the same, that is, both are by the rate of fusion-peptide also result from the action of several HA trimers each undergoing this rate-limiting rearrangement as indicated by the of their individual lag time distributions value of under different proton concentrations (Figure 1). The rate constants derived from gamma distribution fitting of the tlag(pHdrop–arrest) data about to than those for (Figure that the in each arrest and hemifusion, is by different A simple interpretation of these is that virion arrest results from fusion-peptide release and irreversible target-membrane insertion from several HA trimers in the of virus-target membrane contact and that hemifusion is by the same HA fusion-peptide release and membrane but requires a for the that is, their to each other, and is thus to a different of molecular events in the contact between virion and target membrane To the of our and to probe the relationship between the value of N derived from gamma-distribution fitting of hemifusion lag times and the actual number of HA trimers in the hemifusion we the following computer (Figure We a contact of various between 50 and HAs as a of trimers. Our of a contact that can up to about 50 HA trimers, shown in Figure is on of a virion with in et al., 2010). used in our current experiments are on average approximately and et al., see ‘Materials and and thus the actual contact can on average up to about HA trimers. For each virion we obtained lag times for individual HA triggering and

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

BackgroundThe highly pathogenic avian influenza (HPAI) H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA) envelope protein is the primary target for subunit vaccine development.Methodology/Principal FindingsWe used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA) proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a) an inactivated H5N1 vaccine virus, or (b) a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers.Conclusion/SignificanceOur findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.

Binding inhibition of various influenza viruses by sialyllactose-modified trimer DNAs

Molecular Structures of Native HA Trimers on 2009 H1N1 Pandemic Influenza Virus Complexed with Neutralizing Antibodies

Influenza A virus (IAV) remains an important human pathogen largely because of antigenic drift, the rapid emergence of antibody escape mutants that precludes durable vaccination. The most potent neutralizing antibodies interact with cognate epitopes in the globular "head" domain of hemagglutinin (HA), a homotrimeric glycoprotein. The H1 HA possesses five distinct regions defined by a large number of mouse monoclonal antibodies (MAbs), i.e., Ca1, Ca2, Cb, Sa, and Sb. Ca1-Ca2 sites require HA trimerization to attain full antigenicity, consistent with their locations on opposite sides of the trimer interface. Here, we show that full antigenicity of Cb and Sa sites also requires HA trimerization, as revealed by immunofluorescence microscopy of IAV-infected cells and biochemically by pulse-chase radiolabeling experiments. Surprisingly, epitope antigenicity acquired by HA trimerization persists following acid triggering of the globular domains dissociation and even after proteolytic release of monomeric heads from acid-treated HA. Thus, the requirement for HA trimerization by trimer-specific MAbs mapping to the Ca, Cb, and Sa sites is not dependent upon the bridging of adjacent monomers in the native HA trimer. Rather, complete antigenicity of HA (and, by inference, immunogenicity) requires a final folding step that accompanies its trimerization. Once this conformational change occurs, HA trimers themselves would not necessarily be required to induce a highly diverse neutralizing response to epitopes in the globular domain.

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.

Morphological characterization of a plant-made virus-like particle vaccine bearing influenza virus hemagglutinins by electron microscopy

Conflicting reports in leading journals have indicated the minimum number of influenza hemagglutinin (HA) trimers required for fusion to be between one and eight. Interestingly, the data in these reports are either almost identical, or can be transformed to be directly comparable. Different statistical or phenomenological models, however, were used to analyze these data, resulting in the varied interpretations. In an attempt to resolve this contradiction, we use PABM, a brane calculus we recently introduced, enabling an algorithmic systems biology approach that allows the problem to be modeled in a manner following a biological logic. Since a scalable PABM executor is still under development, we sufficiently simplified the fusion model and analyzed it using the model checker, PRISM. We validated the model against older HA-expressing cell-to-cell fusion data using the same parameters with the exception of three, namely HA and sialic acid (SA) surface densities and the aggregation rate, which were expected to be different as a result of the difference in the experimental setup. Results are consistent with the interpretation that a minimum aggregate size of six HA trimers, of which three undergo a conformational change to become fusogenic, is required for fusion. Of these three, two are free, while one is bound. Finally, we determined the effects of varying the SA surface density and showed that only a limited range of densities permit fusion. Our results demonstrate the potential of modeling in providing more precise interpretations of data.

In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the "head" domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C-terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines.

Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.DOI: http://dx.doi.org/10.7554/eLife.00333.001

Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.

Influenza viruses are enveloped, negative-sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80 to 90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of purified receptor-free and receptor-bound HA, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryoelectron tomography to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even interglycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and the formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8 to 10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

Influenza viruses are enveloped, negative sense single-stranded RNA viruses covered in a dense layer of glycoproteins. Hemagglutinin (HA) accounts for 80-90% of influenza glycoprotein and plays a role in host cell binding and membrane fusion. While previous studies have characterized structures of receptor-free and receptor-bound HA in vitro, the effect of receptor binding on HA organization and structure on virions remains unknown. Here, we used cryo-electron tomography (cryoET) to visualize influenza virions bound to a sialic acid receptor mimic. Overall, receptor binding did not result in significant changes in viral morphology; however, we observed rearrangements of HA trimer organization and orientation. Compared to the even inter-glycoprotein spacing of unliganded HA trimers, receptor binding promotes HA trimer clustering and formation of a triplet of trimers. Subtomogram averaging and refinement yielded 8-10 Å reconstructions that allowed us to visualize specific contacts between HAs from neighboring trimers and identify molecular features that mediate clustering. Taken together, we present new structural evidence that receptor binding triggers clustering of HA trimers, revealing an additional layer of HA dynamics and plasticity.

The hemagglutinin (HA) glycoprotein of influenza virus binds host cell receptors and mediates viral entry. Here we present cryo-EM structures of fully glycosylated HAs from H5N1 and H5N8 influenza viruses. We find that the H5N1 HA can form filaments that comprise two head-to-head HA trimers. Multivalent interactions between the two HA trimers are mediated by glycans attached to N158. The distal Sia1-Gal2-NAG3 sugar moiety of N158 interacts with the receptor binding site on the opposing HA trimer. Additional interactions are observed between NAG3 and residues K222 and K193. The H5N8 HA lacks the N158 glycosylation site and does not form the filamentous structure. However, the H5N8 HA exhibits an auto-inhibition conformation, where the receptor binding site is occupied by the glycan chain attached to residue N169 from a neighboring protomer. These structures represent native HA-glycan interactions, which may closely mimic the receptor-HA interactions on the cell surface.