Influenza Virus Membrane Research Articles

Enhanced HPV16 E6/E7+ tumor eradication via induction of tumor-specific T cells by therapeutic vaccination with virosomes presenting synthetic long peptides

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

Influenza Virus Membrane Fusion Is Promoted by the Endosome-Resident Phospholipid Bis(monoacylglycero)phosphate.

The phospholipid bis(monoacylglycero)phosphate (BMP) is enriched in late endosomal and endolysosomal membranes and is believed to be involved in membrane deformation and generation of intralumenal vesicles within late endosomes. Previous studies have demonstrated that BMP promotes membrane fusion of several enveloped viruses, but a limited effect has been found on influenza virus. Here, we report the use of single-virus fusion assays to dissect BMP's effect on influenza virus fusion in greater depth. In agreement with prior reports, we found that hemifusion kinetics and efficiency were unaffected by the addition of 10-20 mol % BMP to the target membrane. However, using an assay for fusion pore formation and genome exposure, we found full fusion efficiency to be substantially enhanced by the addition of 10-20 mol % BMP to the target membrane, while the kinetics remained unaffected. By comparing BMP to other negatively charged phospholipids, we found the effect on fusion efficiency mainly attributable to headgroup charge, although we also hypothesize a role for BMP's unusual chemical structure. Our results suggest that BMP function as a permissive factor for a wider range of viruses than previously reported. We hypothesize that BMP may be a general cofactor for endosomal entry of enveloped viruses.

Proteolytic activation of SARS-CoV-2 spike protein.

Spike (S) protein cleavage is a crucial step in coronavirus infection. In this review, this process is discussed, with particular focus on the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Compared with influenza virus and paramyxovirus membrane fusion proteins, the cleavage activation mechanism of coronavirus S protein is much more complex. The S protein has two cleavage sites (S1/S2 and S2′), and the cleavage motif for furin protease at the S1/S2 site that results from a unique four‐amino acid insertion is one of the distinguishing features of SARS‐CoV‐2. The viral particle incorporates the S protein, which has already undergone S1/S2 cleavage by furin, and then undergoes further cleavage at the S2′ site, mediated by the type II transmembrane serine protease transmembrane protease serine 2 (TMPRSS2), after binding to the receptor angiotensin‐converting enzyme 2 (ACE2) to facilitate membrane fusion at the plasma membrane. In addition, SARS‐CoV‐2 can enter the cell by endocytosis and be proteolytically activated by cathepsin L, although this is not a major mode of SARS‐CoV‐2 infection. SARS‐CoV‐2 variants with enhanced infectivity have been emerging throughout the ongoing pandemic, and there is a close relationship between enhanced infectivity and changes in S protein cleavability. All four variants of concern carry the D614G mutation, which indirectly enhances S1/S2 cleavability by furin. The P681R mutation of the delta variant directly increases S1/S2 cleavability, enhancing membrane fusion and SARS‐CoV‐2 virulence. Changes in S protein cleavability can significantly impact viral infectivity, tissue tropism, and virulence. Understanding these mechanisms is critical to counteracting the coronavirus pandemic.

Design, synthesis and anti-influenza virus activity of furan-substituted spirothiazolidinones

A new series of N-(3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)carboxamides have been designed, synthesized and evaluated as antiviral agents. The compounds were prepared by condensation of 2-methylfuran-3-carbohydrazide, appropriate carbonyl compounds and sulfanyl acids. The new molecules were characterized by IR, 1H NMR, 13C NMR, mass spectrometry and elemental analysis. Six analogues proved to be active against influenza A/H3N2 virus, the two most protent analogues, 3c and 3d, having an EC50 value of about 1 µM. These findings help to define the SAR of spirothiazolidinone-based inhibitors of the influenza virus membrane fusion process.

Glycan binding and specificity of viral influenza neuraminidases by classical molecular dynamics and replica exchange molecular dynamics simulations

Two important glycoproteins on the influenza virus membrane, hemagglutinin (HA) and neuraminidase (NA), are relevant to virus replication. As previously reported, HA has a substrate specificity towards SIA-2,3-GAL-1,4-NAG (3SL) and SIA-2,6-GAL-1,4-NAG (6SL) glycans, while NA can cleave both types of linkages. However, the substrate binding into NA and its preference are not well understood. In this work, the glycan binding and specificity of human and avian NAs were evaluated by classical molecular dynamics (MD) simulations, whilst the conformational diversity of 3SL avian and 6SL human glycans in an unbound state was investigated by replica exchange MD simulations. The results indicated that the 3SL avian receptor fits well in the binding cavity of all NAs and does not require a conformational change for such binding compared to the flexible shape of the 6SL human receptor. From the QM/MM-GBSA binding free energy and decomposition free energy data, 6SL showed a much stronger binding towards human NAs (H1N1, H2N2 and H3N2) than to avian NAs (H5N1 and H7N9). This suggests that influenza NAs have a substrate specificity corresponding to their HA, indicating the functional balance between the two important glycoproteins. Both linkages show distinct glycan topologies when complexed with NAs, while the flexibility of torsion angles between GAL and NAG in 6SL results in the various shapes of glycan and different binding patterns. Lower conformational diversities of both glycans when bound to NA compared to the unbound state were found, and were required in order to be accommodated within the NA cavity.Communicated by Ramaswamy H. Sarma

Lipid-dependence of target membrane stability during influenza viral fusion

Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed ∼30-87% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately -0.20 nm-1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity.

Influenza Virus-Liposome Fusion Studies Using Fluorescence Dequenching and Cryo-electron Tomography.

Influenza virus enters host cells by fusion of viral and endosomal membranes mediated by the influenza hemagglutinin (HA). The pathway of HA-catalyzed fusion has been widely investigated in influenza virus membrane fusion with liposomes. In this chapter we describe methodology for studying the virus-liposome fusion system using a combination of fluorescence dequenching assays and cryo-electron tomography (cryo-ET). In particular, the fluorescence dequenching is used to monitor the efficiency of membrane fusion between whole influenza viruses labeled with a lipophilic dye (DiD) in the membrane and liposomes labeled with a water-soluble dye (sulforhodamine B). By simultaneously monitoring the two fluorescent signals, we can determine the relative time scales of liposomal content leakage or transfer vs. lipid merging. In addition, cryo-ET offers a means of imaging three-dimensional snapshots of different stages of virus-liposome fusion such as prefusion, fusion intermediates, and postfusion.

Membrane remodeling by the M2 amphipathic helix drives influenza virus membrane scission

Membrane scission is a crucial step in all budding processes, from endocytosis to viral budding. Many proteins have been associated with scission, though the underlying molecular details of how scission is accomplished often remain unknown. Here, we investigate the process of M2-mediated membrane scission during the budding of influenza viruses. Residues 50–61 of the viral M2 protein bind membrane and form an amphipathic α-helix (AH). Membrane binding requires hydrophobic interactions with the lipid tails but not charged interactions with the lipid headgroups. Upon binding, the M2AH induces membrane curvature and lipid ordering, constricting and destabilizing the membrane neck, causing scission. We further show that AHs in the cellular proteins Arf1 and Epsin1 behave in a similar manner. Together, they represent a class of membrane-induced AH domains that alter membrane curvature and fluidity, mediating the scission of constricted membrane necks in multiple biological pathways.

Hemagglutinin Palmitoylation Contributes to Membrane Curvature in Influenza a Virus Assembly and Membrane Fusion

Three cysteine residues in the cytoplasmic tail of influenza virus glycoprotein hemagglutinin (HA) are covalently modified by three fatty acids and highly conserved among HA subtypes. The importance of these S-acylation post-translational modifications is highlighted by a strain-dependence to their role in virus replication either in assembly or in fusion, but the mechanisms by which the modifications exerts any effect are unknown. We studied the effects of HA acylation on influenza virus-like particle (VLP) morphology, glycoprotein spacing, protein incorporation, HA induced curvature, and membrane fusion using cryo-electron tomography (cET), VLP-cell and cell-cell fusion assays, and molecular dynamics. Acylation has a significant effect on VLP envelope curvature but is not a determinant of either VLP morphology or HA lateral spacing. De-acylated mutant HA is correlated with a flatter envelope curvature of the released particles in the absence of the M1 layer compared to wild type HA. The de-acylated mutant HA failed to incorporate an M1 layer within spherical VLP consistent with altered HA-M1 interactions. In cell-cell fusion assays, fusion pore enlargement was not observed, regardless of which strain of influenza was de-acylated (H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72)), suggesting that the role of acylation in membrane fusion is viral strain independent. Fusion without pore enlargement could be partially rescued by the expression of M1 and M2 proteins. The spontaneous curvature of palmitate was calculated by molecular dynamic simulations, and was found to be comparable to curvature values derived from VLP size distributions. Our studies indicate that HA acylation is important for both influenza virus assembly and membrane fusion by controlling membrane curvature and modifying HA's interactions with M1.

Effects of Sterol Substitution on Influenza Viral Membrane Fusion

Influenza virus membranes are enriched in cholesterol in comparison to the plasma membrane, as cholesterol can comprise as much as 50% of viral envelope lipids. This enrichment in sterol content likely plays an adaptive role for the virus. Prior work has shown that influenza viral fusion rates vary with cholesterol concentration of both viral and target membranes. To understand which cholesterol properties affect influenza fusion, we measured fusion kinetics between viral and liposomal membranes where cholesterol was replaced with different sterols. Influenza fusion rates are surprisingly invariant across different sterols in the target membrane (all tested at the same mol %) and across a fairly broad range of sterols in the viral membrane. Two sterols--lanosterol and 25-hydroxycholesterol—did slow fusion moderately when introduced into the viral membrane, while substitution of cholesterol with polar sterols greatly slowed fusion. No other sterols tested showed a significant perturbation in fusion rates, including ones altering membrane phase behavior. Our findings suggest that the rate-limiting step in influenza viral fusion may be sensitive to membrane bending moduli but does not depend strongly on sterol-mediated changes to liquid-liquid phase coexistence in membranes.

Computational and Experimental Studies of Lipid-Protein Interactions in Biomemrane Function

Integral membrane proteins constitute more than 30% of the human genome. Substantial lipid-protein interactions contribute to the structure and function of membrane proteins such as GPCRs and viral membrane ion channels. We address how physical properties of the membrane as well as positive charge on the lipid head group modulate protein activation. The flexible surface model (FSM) describes physical properties of the lipid environment that effect membrane protein activation [3]. According to the FSM, elastic coupling of the membrane lipids to integral membrane proteins define lipid-protein interactions through a balance of curvature and hydrophobic forces. Using UV-visible spectroscopy, FTIR spectroscopy [2], and molecular dynamics (MD) simulations, we discovered that lipid bilayer properties like head group and chain length modulate membrane protein functioning by influencing the conformational states. Using rhodopsin, a canonical G-protein-coupled receptor (GPCR) as a prototype, we show the effect of positively charged membrane lipid head group on rhodopsin activation using UV-visible spectroscopy and MD simulations. We discovered rhodopsin reconstituted in membrane lipids with a positively charged head group like DOTAP shifts the equilibrium to active Meta-II state. We propose a mechanism where membrane lipids with positively charged head group interact with Glu134 present on lipid-protein interface breaking the salt bridge with Arg135 in the conserved ERY motif. The interaction between Glu134 and the positively charged head group stabilizes the active Meta-II state. Our study gives insight on the role of membrane lipid-protein interactions in rhodopsin activation mechanism which can ultimately be extended to influenza virus membrane ion channels and other integral proteins. [1] A.V. Struts et al. (2015) Meth. Mol. Biol. 1271, 133-158. [2] E. Zaitseva et al. (2010) JACS 132, 4815-4821. [3] M.F. Brown (2012) Biochemistry 51, 9782-9795.

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level.

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

Entry properties and entry inhibitors of a human H7N9 influenza virus.

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC50 of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.

Computational investigation of interaction mechanisms between juglone and influenza virus surface glycoproteins

Surface glycoproteins of influenza virus [haemagglutinin (HA) and neuraminidase (NA)] are vital target proteins in current rational drug designs. Here, the molecular recognitions between juglone and A/H5N1 influenza virus membrane glycoproteins were studied through flexible docking and molecular dynamic simulations. The results revealed that juglone has the binding specificity to HA (H5) and NA (N1), especially the spatial match of 2-cyclohexene-1,4-dione ring. N1 rather than H5 protein is responsible for the binding, with the interaction energies of − 72.48 and − 41.91 kcal mol− 1, respectively. The residues Arg152, Arg156, Glu276, Glu277 and Arg292 of N1 protein had important roles during the binding process. Compared with other NA inhibitors, juglone is a potential source of anti-influenza ingredients, with better interaction energy and relatively smaller size. In addition, this work also pointed out how to effectively modify the functional groups of juglone. We hope that the results will aid our understanding of recognitions involving influenza surface glycoproteins with the phenolic compounds and warrant the experimental aspects to design novel anti-influenza drugs.

Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship and in vitro antiviral activity of a novel indole series

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure–activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.

Construction of a novel system for developing inhibitors of influenza virus neuraminidase by yeast cell surface engineering

Neuraminidase (NA) is a glycoprotein of the influenza virus membrane and is involved in releasing newly made virus from infected cells, and thus it is the main target of the current influenza chemotherapy. Novel inhibitors of NA which is easily mutated are urgently required to be developed because of the recent emergence of oseltamivir‐resistant strains and highly pathogenic avian influenza viruses. Yeast cell surface engineering is one of the useful technologies for screening binding proteins with specific affinity to target molecules. In this study, we attempted to construct the efficient system for developing inhibitors of NA using this technology. The head domain of wild‐type or oseltamivir‐resistant (H275Y) H5N1 NA was displayed on yeast cell surface and their activities were evaluated. These results suggested that the displayed head domain of NAs retained the innate characteristics of NAs and was useful for evaluating inhibition activity[1].

Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates

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.

Cationic influenza virosomes as an adjuvanted delivery system for CTL induction by DNA vaccination

DNA vaccines have emerged as an attractive approach to induce CTL responses against cancer and infectious agents in recent years. Although CTL induction by DNA vaccination would be a valuable strategy for controlling viral infections, increasing the potency of DNA vaccines is mandatory before DNA vaccines can make it to the clinic. In this study, we developed and characterized a new and safe adjuvanted delivery system for DNA vaccination using cationic influenza virosomes (CIV). CIV were produced by reconstitution of detergent-solubilized influenza virus membranes in the presence of cationic lipids. Plasmid DNA (pDNA) mixed with these virosomes was efficiently transfected into cells of a mouse macrophage cell line (RAW-Blue cells). Moreover, the cells were effectively activated as demonstrated by production of an NFκB/AP-1-inducible reporter enzyme. Following three intradermal immunizations, CIV-delivered epitope-encoding pDNA induced equal numbers of IFNγ- and granzyme B-producing T cells than a 10-fold higher dose of naked pDNA. Virosomes without cationic lipids also improved induction of cellular immunity by pDNA but to a significantly lower extent than CIV. These findings suggest that pDNA–CIV complexes could be an efficacious delivery system suitable for CTL induction by DNA vaccination.

Monitoring influenza hemagglutinin and glycan interactions using surface plasmon resonance

Hemagglutinin (HA) is a trimeric glycoprotein expressed on the influenza virus membrane. HA of influenza viruses binds to the host's cell surface complex glycans via a terminal sialic acid (Sia), as the first key step in the process of infection, transmission and virulence of influenza viruses. It is important to monitor and evaluate the receptor (glycan) binding preferences of the HAs derived from influenza A viruses, especially those originating from birds and swine, to understand their potential ability for interspecies transmission. From this viewpoint, in the present study, we have developed a protocol for analyzing the glycan–HA interactions efficiently and kinetically, based on surface plasmon resonance (SPR). Our results showed that glycan–HA binding analyses can be performed reliably and efficiently on Biacore-chips in the SPR system, using chemically synthesized biotinylated multivalent-glycans. Using the CAP-chip, we were able to regenerate the surface for multiple analyses, allowing us to derive, for the first time, the precise kinetic parameters for different HA–glycan complexes of newly emerging influenza viruses. These studies suggested that this SPR-based method is suitable for influenza surveillance to define the pandemic scenario as well as to screen of synthetic glycans and other compounds that may interfere with glycan–HA interactions.

Architecture of a nascent viral fusion pore

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three-dimensional architecture of influenza virus–liposome complexes at pH 5.5 was investigated by electron cryo-tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane-centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of ‘leaky' fusion to the observed prefusion structures is discussed.