Molecular requirements for a pandemic influenza virus: An acid-stable hemagglutinin protein

Influenza pandemics require that a virus containing a hemagglutinin (HA) surface antigen previously unseen by a majority of the population becomes airborne-transmissible between humans. Although the HA protein is central to the emergence of a pandemic influenza virus, its required molecular properties for sustained transmission between humans are poorly defined. During virus entry, the HA protein binds receptors and is triggered by low pH in the endosome to cause membrane fusion; during egress, HA contributes to virus assembly and morphology. In 2009, a swine influenza virus (pH1N1) jumped to humans and spread globally. Here we link the pandemic potential of pH1N1 to its HA acid stability, or the pH at which this one-time-use nanomachine is either triggered to cause fusion or becomes inactivated in the absence of a target membrane. In surveillance isolates, our data show HA activation pH values decreased during the evolution of H1N1 from precursors in swine (pH 5.5–6.0), to early 2009 human cases (pH 5.5), and then to later human isolates (pH 5.2–5.4). A loss-of-function pH1N1 virus with a destabilizing HA1-Y17H mutation (pH 6.0) was less pathogenic in mice and ferrets, less transmissible by contact, and no longer airborne-transmissible. A ferret-adapted revertant (HA1-H17Y/HA2-R106K) regained airborne transmissibility by stabilizing HA to an activation pH of 5.3, similar to that of human-adapted isolates from late 2009–2014. Overall, these studies reveal that a stable HA (activation pH ≤ 5.5) is necessary for pH1N1 influenza virus pathogenicity and airborne transmissibility in ferrets and is associated with pandemic potential in humans.Wild aquatic birds are thought to be the natural reservoir of influenza A viruses (1). Influenza pandemics occur every few decades, and swine are widely believed to be a key factor in the genesis of pandemics by facilitating reassortment of the eight viral gene segments and replacing avian-like (α-2,3-linked) hemagglutinin (HA) sialic acid receptor-binding specificity with human-like (α-2,6-linked) (2). If the molecular adaptations that allow efficient human-to-human transmissibility are understood, then circulating viruses undergoing these changes (i.e., those with the greatest pandemic potential) could be identified.In 2009, pandemic (p) H1N1 emerged from swine and swiftly infected more than 60 million people, causing 12,000 US deaths in the first year (3). The pandemic strain originated by reassortment in swine, combining five genes (PB1, PB2, PA, NP, and NS) from North American triple-reassortant swine (TRS) viruses, two genes (NA and M) from Eurasian avian-like swine viruses, and an HA gene closely related to that of the classical swine lineage (4). pH1N1 viruses continue to circulate as seasonal H1N1 viruses. They retain several known pandemic traits, including α-2,6-linked sialic acid receptor-binding specificity of the HA, functional balance of HA and NA activity, and a polymerase adapted to the mammalian upper airway (5). Although these traits appear to be necessary for airborne transmissibility of influenza viruses, they do not appear to be sufficient. For example, H5N1 viruses engineered to have these traits were not air-transmissible among ferrets until a mutation increased HA thermostability and lowered the HA activation pH (6–8). The importance of HA stabilization in supporting the adaptation of influenza viruses to humans or enabling a human pandemic is not completely understood.After receptor binding and endocytosis, low pH triggers irreversible structural changes in the HA protein that fuse the viral envelope and host endosomal membrane (9). Measured HA activation pH values across all subtypes and species range from ∼5.0 to 6.0, trending higher in avian viruses (pH 5.6–6.0) and lower in human viruses (pH 5.0–5.5) (10).The goal of this study was to define the role of HA acid stability in pH1N1 pandemic capability. Our data show that HA activation pH decreased as H1N1 adapted from swine to humans. Complementary experiments in ferrets recapitulated this evolution, as we observed a loss-of-function pH1N1 virus acquired airborne transmissibility via stabilizing mutations. Overall, these studies link a fundamental molecular property, the barrier for activation of a membrane fusion protein (for influenza virus HA, its acid stability), to the interspecies adaptation of a ubiquitous respiratory virus.     

Playing Hide and Seek: How Glycosylation of the Influenza Virus Hemagglutinin Can Modulate the Immune Response to Infection

Seasonal influenza A viruses (IAV) originate from pandemic IAV and have undergone changes in antigenic structure, including addition of glycans to the hemagglutinin (HA) glycoprotein. The viral HA is the major target recognized by neutralizing antibodies and glycans have been proposed to shield antigenic sites on HA, thereby promoting virus survival in the face of widespread vaccination and/or infection. However, addition of glycans can also interfere with the receptor binding properties of HA and this must be compensated for by additional mutations, creating a fitness barrier to accumulation of glycosylation sites. In addition, glycans on HA are also recognized by phylogenetically ancient lectins of the innate immune system and the benefit provided by evasion of humoral immunity is balanced by attenuation of infection. Therefore, a fine balance must exist regarding the optimal pattern of HA glycosylation to offset competing pressures associated with recognition by innate defenses, evasion of humoral immunity and maintenance of virus fitness. In this review, we examine HA glycosylation patterns of IAV associated with pandemic and seasonal influenza and discuss recent advancements in our understanding of interactions between IAV glycans and components of innate and adaptive immunity.     

Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.     

Amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the pH of glycoprotein transport vesicles.

Mutants of influenza Rostock virus (H7N1 subtype) were selected for resistance to amantadine hydrochloride at concentrations of the antiviral drug known to affect the function of the virus M2 transmembrane protein. Sequence analysis revealed that three mutants had no changes in M2 but contained a lysine to isoleucine substitution in the hemagglutinin (HA) membrane glycoprotein at position 58 of HA2. The mutant viruses were found to fuse membranes at a pH value 0.7 lower than wild type and to exhibit changes in the conformation of their HAs specifically at the lower pH. The homologous lysine to isoleucine substitution was introduced by site-specific mutagenesis into the HA of X-31 influenza virus (H3 subtype), which was expressed by using vaccinia virus recombinants. The expressed HA also mediated membrane fusion and changed in conformation at a pH value 0.7 lower than wild type. These results indicate that increased acid stability of the HA obviates the consequences of the inhibition of M2 function by amantadine and provide further evidence for the role of M2 in regulating the pH of vesicles involved in glycoprotein transport to the cell surface.     

Influenza A Virus Entry Inhibitors Targeting the Hemagglutinin

Influenza A virus (IAV) has caused seasonal influenza epidemics and influenza pandemics, which resulted in serious threat to public health and socioeconomic impacts. Until now, only 5 drugs belong to two categories are used for prophylaxis and treatment of IAV infection. Hemagglutinin (HA), the envelope glycoprotein of IAV, plays a critical role in viral binding, fusion and entry. Therefore, HA is an attractive target for developing anti‑IAV drugs to block the entry step of IAV infection. Here we reviewed the recent progress in the study of conformational changes of HA during viral fusion process and the development of HA-based IAV entry inhibitors, which may provide a new choice for controlling future influenza pandemics.     

Genomic Evidence for Sequestration of Influenza A Virus Lineages in Sea Duck Host Species

Wild birds are considered the natural reservoir of influenza A viruses (IAVs) making them critical for IAV surveillance efforts. While sea ducks have played a role in novel IAV emergence events that threatened food security and public health, very few surveillance samples have been collected from sea duck hosts. From 2014–2018, we conducted surveillance focused in the Mississippi flyway, USA at locations where sea duck harvest has been relatively successful compared to our other sampling locations. Our surveillance yielded 1662 samples from sea ducks, from which we recovered 77 IAV isolates. Our analyses identified persistence of sea duck specific IAV lineages across multiple years. We also recovered sea duck origin IAVs containing an H4 gene highly divergent from the majority of North American H4-HA with clade node age of over 65 years. Identification of IAVs with long branch lengths is indicative of substantial genomic change consistent with persistence without detection by surveillance efforts. Sea ducks play a role in the movement and long-term persistence of IAVs and are likely harboring more undetected IAV diversity. Sea ducks should be a point of emphasis for future North American wild bird IAV surveillance efforts.     

Influenza Virus Infections in Cats

In the past, cats were considered resistant to influenza. Today, we know that they are susceptible to some influenza A viruses (IAVs) originating in other species. Usually, the outcome is only subclinical infection or a mild fever. However, outbreaks of feline disease caused by canine H3N2 IAV with fever, tachypnoea, sneezing, coughing, dyspnoea and lethargy are occasionally noted in shelters. In one such outbreak, the morbidity rate was 100% and the mortality rate was 40%. Recently, avian H7N2 IAV infection occurred in cats in some shelters in the USA, inducing mostly mild respiratory disease. Furthermore, cats are susceptible to experimental infection with the human H3N2 IAV that caused the pandemic in 1968. Several studies indicated that cats worldwide could be infected by H1N1 IAV during the subsequent human pandemic in 2009. In one shelter, severe cases with fatalities were noted. Finally, the highly pathogenic avian H5N1 IAV can induce a severe, fatal disease in cats, and can spread via cat-to-cat contact. In this review, the Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European countries, summarises current data regarding the aetiology, epidemiology, pathogenesis, clinical picture, diagnostics, and control of feline IAV infections, as well as the zoonotic risks.     

Natural Selection of H5N1 Avian Influenza A Viruses with Increased PA-X and NS1 Shutoff Activity

Influenza A viruses (IAV) can infect a broad range of mammalian and avian species. However, the host innate immune system provides defenses that restrict IAV replication and infection. Likewise, IAV have evolved to develop efficient mechanisms to counteract host antiviral responses to efficiently replicate in their hosts. The IAV PA-X and NS1 non-structural proteins are key virulence factors that modulate innate immune responses and virus pathogenicity during infection. To study the determinants of IAV pathogenicity and their functional co-evolution, we evaluated amino acid differences in the PA-X and NS1 proteins of early (1996–1997) and more recent (since 2016) H5N1 IAV. H5N1 IAV have zoonotic and pandemic potential and represent an important challenge both in poultry farming and human health. The results indicate that amino acid changes occurred over time, affecting the ability of these two non-structural H5N1 IAV proteins to inhibit gene expression and affecting virus pathogenicity. These results highlight the importance to monitor the evolution of these two virulence factors of IAV, which could result in enhanced viral replication and virulence.     

Molecular and cellular aspects of rhabdovirus entry

Rhabdoviruses enter the cell via the endocytic pathway and subsequently fuse with a cellular membrane within the acidic environment of the endosome. Both receptor recognition and membrane fusion are mediated by a single transmembrane viral glycoprotein (G). Fusion is triggered via a low-pH induced structural rearrangement. G is an atypical fusion protein as there is a pH-dependent equilibrium between its pre- and post-fusion conformations. The elucidation of the atomic structures of these two conformations for the vesicular stomatitis virus (VSV) G has revealed that it is different from the previously characterized class I and class II fusion proteins. In this review, the pre- and post-fusion VSV G structures are presented in detail demonstrating that G combines the features of the class I and class II fusion proteins. In addition to these similarities, these G structures also reveal some particularities that expand our understanding of the working of fusion machineries. Combined with data from recent studies that revealed the cellular aspects of the initial stages of rhabdovirus infection, all these data give an integrated view of the entry pathway of rhabdoviruses into their host cell.     

A capsid protein of nonenveloped Bluetongue virus exhibits membrane fusion activity

The outer capsid layer of Bluetongue virus, a member of the nonenveloped Reoviridae family, is composed of two proteins, a receptor-binding protein, VP2, and a second protein, VP5, which shares structural features with class I fusion proteins of enveloped viruses. In the replication cycle of Bluetongue virus VP5 acts as a membrane permeabilization protein that mediates release of viral particles from endosomal compartments into the cytoplasm. Here, we show that VP5 can also act as a fusion protein and induce syncytium formation when it is fused to a transmembrane anchor and expressed on the cell surface. Fusion activity is strictly pH-dependent and is triggered by short exposure to low pH. No cell-cell fusion is observed at neutral pH. Deletion of the first 40 amino acids, which can fold into two amphipathic helices, abolishes fusion activity. Syncytium formation by VP5 is inhibited in the presence of VP2 when it is expressed in a membrane-anchored form. The data indicate an interaction between the outer capsid protein VP2 and VP5 and show that VP5 undergoes pH-dependent conformational changes that render it capable of interacting with cellular membranes. More importantly, our data show that a membrane permeabilization protein of a nonenveloped virus can evolve into a fusion protein by the addition of an appropriate transmembrane anchor. The results strongly suggest that the mechanism of membrane permeabilization by VP5 and membrane fusion by viral fusion proteins require similar structural features and conformational changes.     

Insights into bunyavirus architecture from electron cryotomography of Uukuniemi virus

Bunyaviridae is a large family of viruses that have gained attention as "emerging viruses" because many members cause serious disease in humans, with an increasing number of outbreaks. These negative-strand RNA viruses possess a membrane envelope covered by glycoproteins. The virions are pleiomorphic and thus have not been amenable to structural characterization using common techniques that involve averaging of electron microscopic images. Here, we determined the three-dimensional structure of a member of the Bunyaviridae family by using electron cryotomography. The genome, incorporated as a complex with the nucleoprotein inside the virions, was seen as a thread-like structure partially interacting with the viral membrane. Although no ordered nucleocapsid was observed, lateral interactions between the two membrane glycoproteins determine the structure of the viral particles. In the most regular particles, the glycoprotein protrusions, or "spikes," were seen to be arranged on an icosahedral lattice, with T = 12 triangulation. This arrangement has not yet been proven for a virus. Two distinctly different spike conformations were observed, which were shown to depend on pH. This finding is reminiscent of the fusion proteins of alpha-, flavi-, and influenza viruses, in which conformational changes occur in the low pH of the endosome to facilitate fusion of the viral and host membrane during viral entry.     

The Immunomodulatory CEA Cell Adhesion Molecule 6 (CEACAM6/CD66c) Is a Protein Receptor for the Influenza A Virus

To establish a productive infection in host cells, viruses often use one or multiple host membrane glycoproteins as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein, binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found that CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (Carcinoembryonic cell adhesion molecule 6 or CEACAM6) as a glycoprotein receptor for Influenza A virus.     

Dual R108K and G189D Mutations in the NS1 Protein of A/H1N1 Influenza Virus Counteract Host Innate Immune Responses

Influenza A viruses (IAV) modulate host antiviral responses to promote growth and pathogenicity. Here, we examined the multifunctional IAV nonstructural protein 1 (NS1) of influenza A virus to better understand factors that contribute to viral replication efficiency or pathogenicity. In 2009, a pandemic H1N1 IAV (A/California/07/2009 pH1N1) emerged in the human population from swine. Seasonal variants of this virus are still circulating in humans. Here, we compared the sequence of a seasonal variant of this H1N1 influenza virus (A/Urumqi/XJ49/2018(H1N1), first isolated in 2018) with the pandemic strain A/California/07/2009. The 2018 virus harbored amino acid mutations (I123V and N205S) in important functional sites; however, 108R and 189G were highly conserved between A/California/07/2009 and the 2018 variant. To better understand interactions between influenza viruses and the human innate immune system, we generated and rescued seasonal 2009 H1N1 IAV mutants expressing an NS1 protein harboring a dual mutation (R108K/G189D) at these conserved residues and then analyzed its biological characteristics. We found that the mutated NS1 protein exhibited systematic and selective inhibition of cytokine responses via a mechanism that may not involve binding to cleavage and polyadenylation specificity factor 30 (CPSF30). These results highlight the complexity underlying host–influenza NS1 protein interactions.     

Quercetin as an Antiviral Agent Inhibits Influenza A Virus (IAV) Entry

Influenza A viruses (IAVs) cause seasonal pandemics and epidemics with high morbidity and mortality, which calls for effective anti-IAV agents. The glycoprotein hemagglutinin of influenza virus plays a crucial role in the initial stage of virus infection, making it a potential target for anti-influenza therapeutics development. Here we found that quercetin inhibited influenza infection with a wide spectrum of strains, including A/Puerto Rico/8/34 (H1N1), A/FM-1/47/1 (H1N1), and A/Aichi/2/68 (H3N2) with half maximal inhibitory concentration (IC₅₀) of 7.756 ± 1.097, 6.225 ± 0.467, and 2.738 ± 1.931 μg/mL, respectively. Mechanism studies identified that quercetin showed interaction with the HA2 subunit. Moreover, quercetin could inhibit the entry of the H5N1 virus using the pseudovirus-based drug screening system. This study indicates that quercetin showing inhibitory activity in the early stage of influenza infection provides a future therapeutic option to develop effective, safe and affordable natural products for the treatment and prophylaxis of IAV infections.     

The Effects of Genetic Variation on H7N9 Avian Influenza Virus Pathogenicity

Since the H7N9 avian influenza virus emerged in China in 2013, there have been five seasonal waves which have shown human infections and caused high fatality rates in infected patients. A multibasic amino acid insertion seen in the HA of current H7N9 viruses occurred through natural evolution and reassortment, and created a high pathogenicity avian influenza (HPAI) virus from the low pathogenicity avian influenza (LPAI) in 2017, and significantly increased pathogenicity in poultry, resulting in widespread HPAI H7N9 in poultry, which along with LPAI H7N9, contributed to the severe fifth seasonal wave in China. H7N9 is a novel reassorted virus from three different subtypes of influenza A viruses (IAVs) which displays a great potential threat to public health and the poultry industry. To date, no sustained human-to-human transmission has been recorded by the WHO. However, the high ability of evolutionary adaptation of H7N9 and lack of pre-existing immunity in humans heightens the pandemic potential. Changes in IAVs proteins can affect the viral transmissibility, receptor binding specificity, pathogenicity, and virulence. The multibasic amino acid insertion, mutations in hemagglutinin, deletion and mutations in neuraminidase, and mutations in PB2 contribute to different virological characteristics. This review summarized the latest research evidence to describe the impacts of viral protein changes in viral adaptation and pathogenicity of H7N9, aiming to provide better insights for developing and enhancing early warning or intervention strategies with the goal of preventing highly pathogenic IAVs circulation in live poultry, and transmission to humans.     

Avian Influenza A Viruses Reassort and Diversify Differently in Mallards and Mammals

Reassortment among co-infecting influenza A viruses (IAVs) is an important source of viral diversity and can facilitate expansion into novel host species. Indeed, reassortment played a key role in the evolution of the last three pandemic IAVs. Observed patterns of reassortment within a coinfected host are likely to be shaped by several factors, including viral load, the extent of viral mixing within the host and the stringency of selection. These factors in turn are expected to vary among the diverse host species that IAV infects. To investigate host differences in IAV reassortment, here we examined reassortment of two distinct avian IAVs within their natural host (mallards) and a mammalian model system (guinea pigs). Animals were co-inoculated with A/wildbird/California/187718-36/2008 (H3N8) and A/mallard/Colorado/P66F1-5/2008 (H4N6) viruses. Longitudinal samples were collected from the cloaca of mallards or the nasal tract of guinea pigs and viral genetic exchange was monitored by genotyping clonal isolates from these samples. Relative to those in guinea pigs, viral populations in mallards showed higher frequencies of reassortant genotypes and were characterized by higher genotype richness and diversity. In line with these observations, analysis of pairwise segment combinations revealed lower linkage disequilibrium in mallards as compared to guinea pigs. No clear longitudinal patterns in richness, diversity or linkage disequilibrium were present in either host. Our results reveal mallards to be a highly permissive host for IAV reassortment and suggest that reduced viral mixing limits avian IAV reassortment in a mammalian host.     

Influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion

Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future.     

The curious case of arenavirus entry, and its inhibition

Arenaviruses comprise a diverse family of enveloped negative-strand RNA viruses that are endemic to specific rodent hosts worldwide. Several arenaviruses cause severe hemorrhagic fevers in humans, including Junín and Machupo viruses in South America and Lassa fever virus in western Africa. Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins. We will review the curious properties of the tripartite GPC complex and describe evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention.     

Reversible structural changes in the influenza hemagglutinin precursor at membrane fusion pH

The subunits of the influenza hemagglutinin (HA) trimer are synthesized as single-chain precursors (HA0s) that are proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2. Cleavage is required for activation of membrane fusion at low pH, which occurs at the beginning of infection following transfer of cell-surface–bound viruses into endosomes. Activation results in extensive changes in the conformation of cleaved HA. To establish the overall contribution of cleavage to the mechanism of HA-mediated membrane fusion, we used cryogenic electron microscopy (cryo-EM) to directly image HA0 at neutral and low pH. We found extensive pH-induced structural changes, some of which were similar to those described for intermediates in the refolding of cleaved HA at low pH. They involve a partial extension of the long central coiled coil formed by melting of the preexisting secondary structure, threading it between the membrane-distal domains, and subsequent refolding as extended helices. The fusion peptide, covalently linked at its N terminus, adopts an amphipathic helical conformation over part of its length and is repositioned and packed against a complementary surface groove of conserved residues. Furthermore, and in contrast to cleaved HA, the changes in HA0 structure at low pH are reversible on reincubation at neutral pH. We discuss the implications of covalently restricted HA0 refolding for the cleaved HA conformational changes that mediate membrane fusion and for the action of antiviral drug candidates and cross-reactive anti-HA antibodies that can block influenza infectivity.

The membranes of lipid enveloped viruses fuse with cellular membranes at the beginning of infection to deliver their genetic material into cells. For some viruses, fusion is at the cell surface; for others, it occurs following transfer of receptor-bound viruses into endosomes. Influenza viruses are in the second group and the virus glycoprotein involved in both receptor-binding and low-pH–triggered membrane fusion is hemagglutinin (HA). HA is synthesized as a precursor, HA0, that is proteolytically cleaved during virus replication into the two disulphide-linked components of infectious virus hemagglutinin, HA1 and HA2 (1–4). For 14 of the 16 HA subtypes, cleavage is by trypsin-like enzymes (5, 6) at an arginine residue that immediately precedes the N terminus of HA2 (7). For some HA0s of the two remaining subtypes, H5 and H7, the arginine residue at the site of cleavage is part of a furin-recognition sequence (8), the presence of which generally correlates with virus pathogenicity (9–11).The three-dimensional (3D) structures of HA0 and HA, before and after cleavage, differ only near the site of cleavage (12, 13). Nevertheless, cleavage is essential for fusion activity (14, 15). It generates the HA2 N terminus at a conserved hydrophobic sequence, which is called the fusion peptide because its synthetic peptide analogs have membrane fusion activity. It has been envisioned that cleavage and sequestering of the N terminus of the fusion peptide in a conserved pocket primes HA for its response to low pH and that activation of subsequent changes in conformation involves release of the fusion peptide from its buried location (16, 17).We have previously studied conformational changes in cleaved HA following incubation at fusion pH (18) using cryogenic electron microscopy (cryo-EM) and identified intermediates in the process. To investigate the importance of cleavage to the mechanism of HA-mediated membrane fusion, we now study the response of HA0 to incubation at low pH. We find that the HA0 structure is extensively changed at low pH and that, unlike the changes detected in cleaved HA, the changes in HA0 are reversible on reincubation at neutral pH. We compare the structure of HA0 at low pH with its structure at neutral pH and with one of the low-pH cleaved-HA intermediates (state IV), the “extended intermediate.” This particularly prominent intermediate contains a long central trimeric coiled coil that is assumed to deliver the fusion peptides at its N termini to target cell membranes and to form a bridge between the HA-associated virus membrane and the target. We discuss the possibility that, although not directly involved in membrane fusion, the changes in conformation of HA0 at low pH are indicators of early changes in the conformation of cleaved HA that are required for membrane fusion.     

Influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion

Lipid raft microdomains are enriched in sphingomyelin and cholesterol and function as platforms for signal transduction and as the site of budding of several enveloped viruses, including influenza virus. The influenza virus hemagglutinin (HA) glycoprotein, which mediates both viral-cell attachment and membrane fusion, associates intrinsically with lipid rafts. Residues in the HA transmembrane (TM) domain are important for raft association as sequence substitutions in the HA TM domain ablate HA association with rafts (nonraft HA). Cells expressing either WT or nonraft HA cause complete fusion (lipid mixing and content mixing) over widely varying HA expression levels. However, the number of fusion events measured for nonraft HA mutant protein at all HA surface densities was reduced to approximately 55% of the events for WT HA protein. Mutant influenza viruses were generated that contain the nonraft HA TM domain alterations. Electron microscopy experiments showed that WT HA was distributed at the cell surface in clusters of 200-280 nm in diameter, whereas nonraft HA was distributed mostly randomly at the plasma membrane. Nonraft HA virus showed reduced budding, contained reduced amounts of HA protein, was greatly reduced in infectivity, and exhibited decreased virus-membrane fusion activity. Cholesterol depletion of virus did not affect the ability of virions to cause either virus-cell lipid mixing or virus-mediated hemolysis, a surrogate for content mixing. Taken together, the data suggest that HA clusters in rafts to provide a sufficient concentration of HA in budding virus to mediate efficient virus-cell fusion.