Fusion Peptide Domain Research Articles

Enhancing immune protection against MERS-CoV: the synergistic effect of proteolytic cleavage sites and the fusion peptide and RBD domain targeting VLP immunization.

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic infectious virus that has caused significant outbreaks in the Middle East and beyond. Due to a highly mortality rate, easy transmission, and rapid spread of the MERS-CoV, it remains as a significant public health treat. There is currently no licensed vaccine available to protect against MERS-CoV. In this study, we investigated whether the proteolytic cleavage sites and fusion peptide domain of the MERS-CoV spike (S) protein could be a vaccine target to elicit the MERS-CoV S protein-specific antibody responses and confer immune protection against MERS-CoV infection. Our results demonstrate that immunization of the proteolytic cleavage sites and the fusion peptide domain using virus-like particle (VLP) induced the MERS-CoV S protein-specific IgG antibodies with capacity to neutralize pseudotyped MERS-CoV infection in vitro. Moreover, proteolytic cleavage sites and the fusion peptide VLP immunization showed a synergistic effect on the immune protection against MERS-CoV infection elicited by immunization with VLP expressing the receptor binding domain (RBD) of the S protein. Additionally, immune evasion of MERS-CoV RBD variants from anti-RBD sera was significantly controlled by anti-proteolytic cleavage sites and the fusion peptide sera. Our study demonstrates the potential of VLP immunization targeting the proteolytic cleavage sites and the fusion peptide and RBD domains of the MERS-CoV S protein for the development of effective treatments and vaccines against MERS-CoV and related variants.

Pathway of pore formation by influenza fusion peptides investigated in atomistic detail.

The fusion peptide (FP) domain is responsible for the fusogenic activity of spike glycoproteins in a variety of enveloped viruses. As the only portion of the protein that interacts directly with the target membrane lipid tails, this domain is integral to the virus’ ability to infect the host cell. Additionally, experiments have consistently demonstrated the ability of the fusion peptide domain to porate or rupture model membrane systems. Here, we present the results of over 120 μs of atomistic molecular dynamics simulations investigating the pathway by which FP-induced pores form in membranes composed of lysolipid and POPC. Both isolated FP and FP aggregates spontaneously form pores in these membranes on hundred-ns time scales. Pores formed by isolated FP are generated in the lipid portion of the membrane, rather than under the peptide, consistent with the classical view of peptides as membrane components that alter the average properties of the leaflet. Peptide aggregates, on the other hand, mostly form pores via peptide-pore intermediates, where the cis leaflet is spanned primarily by highly tilted FP, locally reducing the hydrophobic thickness that must be bridged by a water wire or hydrophobic defect. By restraining a single FP within an FP aggregate to this tilted conformation, pores form in lower lysolipid-content membranes, including pure POPC, through a similar pathway.

Initiation and evolution of pores formed by influenza fusion peptides probed by lysolipid inclusion

The fusion peptide (FP) domain is necessary for the fusogenic activity of spike proteins in a variety of enveloped viruses, allowing the virus to infect the host cell, and is the only part of the protein that interacts directly with the target membrane lipid tails during fusion. There are consistent findings of poration by this domain in experimental model membrane systems, and, in certain conditions, the isolated FPs can generate pores. Here, we use molecular dynamics simulations to investigate the specifics of how these FP-induced pores form in membranes with different compositions of lysolipid and POPC. The simulations show that pores form spontaneously at high lysolipid concentrations via hybrid intermediates, where FP aggregates in the cis leaflet tilt to form a funnel-like structure that spans the leaflet and locally reduces the hydrophobic thickness that must be traversed by water to form a pore. By restraining a single FP within an FP aggregate to this tilted conformation, pores can be formed in lower-lysolipid-content membranes, including pure POPC, on the 100-ns timescale, much more rapidly than in unbiased simulations in bilayers with the same composition. The pore formation pathway is similar to the spontaneous formation in high lysolipid concentrations. Depending on the membrane composition, the pores can be metastable (as seen in POPC) or lead to membrane rupture.

Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)–reactive and SARS-CoV-2–cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830–reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti–SARS-CoV-2-S1-IgG antibodies. Spike–cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike–cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Discovery of Small Molecule Entry Inhibitors Targeting the Fusion Peptide of SARS-CoV-2 Spike Protein.

SARS-CoV-2 entry into host cells relies on the spike (S) protein binding to the human ACE2 receptor. In this study, we investigated the structural dynamics of the viral S protein at the fusion peptide (FP) domain and small molecule binding for therapeutics development. Following comparative modeling analysis and docking studies of our previously identified fusion inhibitor chlorcyclizine, we performed a pharmacophore-based virtual screen and identified two novel chemotypes of entry inhibitors targeting the FP. The compounds were evaluated in the pseudoparticle viral entry assay and SARS-CoV-2 cytopathic effect assay and showed single-digital micromole inhibition against SARS-CoV-2 as well as SARS-CoV-1 and MERS. The characterization of the FP binding site of SARS-CoV-2 S protein provides a promising target for the structure-based development of small molecule entry inhibitors as drug candidates for the treatment of COVID-19.

Membranotropic and biological activities of the membrane fusion peptides from SARS-CoV spike glycoprotein: The importance of the complete internal fusion peptide domain.

Fusion peptides (FP) are prominent hydrophobic segments of viral fusion proteins that play critical roles in viral entry. FPs interact with and insert into the host lipid membranes, triggering conformational changes in the viral protein that leads to the viral-cell fusion. Multiple membrane-active domains from the severe acute respiratory syndrome (SARS) coronavirus (CoV) spike protein have been reported to act as the functional fusion peptide such as the peptide sequence located between the S1/S2 and S2’ cleavage sites (FP1), the S2’-adjacent fusion peptide domain (FP2), and the internal FP sequence (cIFP). Using a combined biophysical approach, we demonstrated that the α-helical coiled-coil-forming internal cIFP displayed the highest membrane fusion and permeabilizing activities along with membrane ordering effect in phosphatidylcholine (PC)/phosphatidylglycerol (PG) unilamellar vesicles compared to the other two N-proximal fusion peptide counterparts. While the FP1 sequence displayed intermediate membranotropic activities, the well-conserved FP2 peptide was substantially less effective in promoting fusion, leakage, and membrane ordering in PC/PG model membranes. Furthermore, Ca2+ did not enhance the FP2-induced lipid mixing activity in PC/phosphatidylserine/cholesterol lipid membranes, despite its strong erythrocyte membrane perturbation. Nonetheless, we found that the three putative SARS-CoV membrane-active fusion peptide sequences here studied altered the physical properties of model and erythrocyte membranes to different extents. The importance of the distinct membranotropic and biological activities of all SARS-CoV fusion peptide domains and the pronounced effect of the internal fusion peptide sequence to the whole spike-mediated membrane fusion process are discussed.

In-silico Study to elucidate corona Virus by plant phytoderivatives that hits as a fusion inhibitors targeting HR1 domain in spike protein which conformational Changes efficiently inhibit entry COVID-19

Introduction: COVID-19 could be a human beta corona virus that have potential source of severe widespread respiratory and asymptomatic multiple pathophysiological conditions and is belonging to the SARS and MERS β-corona viruses linage that have inflated mortality rates and acute potential of pandemic. The viral envelope surface spike glycoprotein (S) binding with host cell receptor angiotensin-converting enzyme 2 (ACE2) and conciliate fuse the virus particle inside the host cell membranes, promising spike protein substantially important to endocytosis and host species an involuntary orienting response. Methods: Within the present in-silico study, two plant bioactive compounds namely ALS-1 and ALS-2 (from Alangium salvifolium) were analyzed for his or her inhibitory role on fusion peptide region or S2 HR-1 domain and efficiently block virus entry into host cell by applying the molecular simulation, docking studies. Other parameters viz. determination of molecular interaction-based binding affinity values, protein-ligand interactions, Lipinski rule of 5, functional properties and biological activities for the above compounds were also calculated by employing the acceptable bioinformatics tools. Results: The results of docking analysis clearly showed that ALS-1 has highest binding affinity with trimeric Spike glycoprotein (-11.6 kcal/mole) and ALS-2 (-10.8 kcal/mole). Based on protein interaction analysis both phytoderivatives bind HR-1 (fusion peptide) domain. Other parametric results showed good absorption activity and not violated Lipinski score of drug-likeness. Conclusion: Therefore studied plant derivatives may have the potential to play a big role as 2019 n-CoV fusion peptide inhibitor, revealing influential inhibitory activity against S-participated endocytosis and 2019 n-CoV viral infection, suggesting further optimizations (3-DQASR) and pharmaceutical development of both derivatives, respectively, to stop and treat novel COVID-19 infection.

Fully hydrophobic HIV gp41 adopts a hemifusion-like conformation in phospholipid bilayers

The HIV envelope glycoprotein mediates virus entry into target cells by fusing the virus lipid envelope with the cell membrane. This process requires large-scale conformational changes of the fusion protein gp41. Current understanding of the mechanisms with which gp41 induces membrane merger is limited by the fact that the hydrophobic N-terminal fusion peptide (FP) and C-terminal transmembrane domain (TMD) of the protein are challenging to characterize structurally in the lipid bilayer. Here we have expressed a gp41 construct that contains both termini, including the FP, the fusion peptide-proximal region (FPPR), the membrane-proximal external region (MPER), and the TMD. These hydrophobic domains are linked together by a shortened water-soluble ectodomain. We reconstituted this "short NC" gp41 into a virus-mimetic lipid membrane and conducted solid-state NMR experiments to probe the membrane-bound conformation and topology of the protein. 13C chemical shifts indicate that the C-terminal MPER-TMD is predominantly α-helical, whereas the N-terminal FP-FPPR exhibits β-sheet character. Water and lipid 1H polarization transfer to the protein revealed that the TMD is well-inserted into the lipid bilayer, whereas the FPPR and MPER are exposed to the membrane surface. Importantly, correlation signals between the FP-FPPR and the MPER are observed, providing evidence that the ectodomain is sufficiently collapsed to bring the N- and C-terminal hydrophobic domains into close proximity. These results support a hemifusion-like model of the short NC gp41 in which the ectodomain forms a partially folded hairpin that places the FPPR and MPER on the opposing surfaces of two lipid membranes.

Hydrogen-Deuterium Exchange Supports Independent Membrane-Interfacial Fusion Peptide and Transmembrane Domains in Subunit 2 of Influenza Virus Hemagglutinin Protein, a Structured and Aqueous-Protected Connection between the Fusion Peptide and Soluble Ectodomain, and the Importance of Membrane Apposition by the Trimer-of-Hairpins Structure.

The influenza virus hemagglutinin (HA) protein has HA1 and HA2 subunits, which form an initial complex. HA1's bind host cell sialic acids, which triggers endocytosis, HA1/HA2 separation, and HA2-mediated fusion between virus and endosome membranes. We report hydrogen-deuterium exchange mass spectrometry (HDX-MS) on the HA2 subunit without HA1. HA2 contains the fusion peptide (FP), soluble ectodomain (SE), transmembrane domain (TM), and endodomain. FP is a monomer by itself, while SE is a trimer of hairpins that includes an interior bundle of residue 38-105 helices, turns, and residue 154-178 strands packed antiparallel to the bundle. FP and TM extend from the same side of the SE hairpin, and fusion models often depict a FP/TM complex with membrane traversal of both domains that is important for membrane pore expansion. The HDX-MS data of this study do not support this complex and instead support independent FP and TM with respective membrane-interfacial and traversal locations. The data also show a low level of aqueous exposure of the 22-38 segment, consistent with retention of the 23-35 antiparallel β sheet observed in the initial HA1/HA2 complex. We propose the β sheet as a semirigid connector between FP and SE that enables close membrane apposition prior to fusion. The I173E mutant exhibits greater exchange for residues 22-69 and 150-191, consistent with dissociation of SE C-terminal strands from interior N-helices. Similar trends are observed for the G1E mutant as well as less exchange for G1E FP. Fusion is highly impaired with either mutant, which correlates with reduced membrane apposition and, for G1E, FP binding to SE rather than the target membrane.

Peptide Assembly on the Membrane Determines the HIV-1 Inhibitory Activity of Dual-Targeting Fusion Inhibitor Peptides

Novel strategies in the design of HIV-1 fusion/entry inhibitors are based on the construction of dual-targeting fusion proteins and peptides with synergistic antiviral effects. In this work we describe the design of dual-targeting peptides composed of peptide domains of E2 and E1 envelope proteins from Human Pegivirus with the aim of targeting both the loop region and the fusion peptide domains of HIV-1 gp41. In a previous work, we described the inhibitory role of a highly conserved fragment of the E1 protein (domain 139–156) which interacts with the HIV-1 fusion peptide at the membrane level. Here, two different dual-targeting peptides, where this E1 peptide is located on the N- or the C-terminus respectively, have been chemically synthesized and their antiviral activities have been evaluated with HIV pseudotyped viruses from different clades. The study of the functional behaviour of peptides in a membranous environment attending to the peptide recognition of the target sites on gp41, the peptide conformation as well as the peptide affinity to the membrane, demonstrate that antiviral activity of the dual-targeting peptides is directly related to the peptide affinity and its subsequent assembly into the model membrane. The overall results point out to the necessity that fusion inhibitor peptides that specifically interfere with the N-terminal region of gp41 are embedded within the membrane in order to properly interact with their viral target.

PredictFP2: A New Computational Model to Predict Fusion Peptide Domain in All Retroviruses.

Fusion peptide (FP) is a pivotal domain for the entry of retrovirus into host cells to continue self-replication. The crucial role indicates that FP is a promising drug target for therapeutic intervention. A FP model proposed in our previous work is relatively not efficient to predict FP in retroviruses. Thus in this work, we come up with a new computational model to predict FP domains in all the retroviruses. It basically predicts FP domains through recognizing their start and end sites separately with SVM method combing the hydrophobicity knowledge of the subdomain around furin cleavage site. The classification accuracy rates are 91.91, 91.20 and 89.13 percent respectively corresponding to jack-knife, 10-fold cross-validation and 5-fold cross-validation test. Second, this model discovered 69,753 and 493 putative FPs after scanning amino acid sequences and HERV DNA sequences both without FP annotations. Subsequently, a statistical analysis was performed on the 69,753 putative FP sequences, which confirms that FP is a hydrophobic domain. Lastly, we depicted the distribution of the 493 putative FP sequences on each human chromosome and each HERV family, which shows that FP of HERV probably has chromosome and family preference.

The Role of Fusion Protein F in the Virus Entry and Cell‐to‐Cell Spread for Nipah Virus

Viruses are obligate parasites, where they require host cells to replicate and produce their progeny. Membrane and non‐segmented single‐stranded negative sense RNA genome are important signatures of paramyxoviruses. The paramyxovirus family contains many common human pathogenic viruses, including measles, mumps, respiratory syncytial virus, and Nipah virus (NiV) ‐ a deadly paramyxovirus with up to 75% mortality rate, with neither vaccine nor therapeutics available. Membrane fusion is necessary for entry of the membraned viruses like paramyxoviruses into their host cells (viral‐cell fusion), and for cell‐cell virus spread via syncytia formation (cell‐cell fusion). For most paramyxoviruses membrane fusion requires two viral glycoproteins – the attachment glycoprotein (HN/H/G) and fusion (F) glycoprotein. Structurally, type 1 fusion protein and a type 2 attachment protein is common to all paramyxoviruses. For virus entry into host cells during infection or cell‐cell spread, NiV has a unique pair of G and F proteins as its specific attachment glycoprotein and fusion glycoprotein respectively. NiV G protein made up of C‐terminal end globular head, stalk region, transmembrane (TM) domain and N‐terminal cytoplasmic tail. In contrast, NiV F protein consist of N‐terminal tail on the surface, hydrophobic fusion peptide (FP), single‐pass transmembrane (TM) domain, and C‐terminal cytoplasmic tail. It also has two heptad repeat domains (HR1/HRA [closer to FP] and HR2/HRB [closer to TM]). Both FP and HR domains play key roles in membrane fusion. NiV F proteins are synthesized as biologically inactive F0 precursors. To be active, F0 proteins need to be cleaved into metastable state of F1 and F2 fragments, covalently linked by disulphide bonds. Unlike other paramyxoviruses, where the cleaving of F0 into F1 + F2 is mediated by trypsin or trypsin‐like proteases, NiV F is cleaved by endocytosis and pH dependent process by endosomal proteases (cathepsin L/B) which exposes the FP as new N‐terminal of the F1 fragment. On the surface of NiV membrane, metastable structure of F1 + F2 makes the FP ‘hidden’ or ‘protected’ by protein‐protein interaction with G protein. Upon receptor (Ephrin B2/B3) binding, the NiV G protein alters its structure which then nudges and triggers the adjacent F protein to undergo conformational changes that causes the FP to stab into the host cell membrane, thus bridging between virus membrane and host cell membrane. On further conformational changes of F1+F2, the HRA/HR1 and HRB/HR2 coalesce or conjoin which then forms ‘fusion pores' allowing the NiV genome to enter the host cell cytoplasm and begin replication steps. When many similar NiV F and G mediated membrane fusion steps occur between a NiV‐infected cell with the non‐infected neighboring cells, it produces a multi‐nucleated giant syncytia formation, i.e. to facilitate cell‐cell virus spread. The Walton High School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function of NiV F and G proteins in membrane fusion.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Efficient Fusion at Neutral pH by Human Immunodeficiency Virus gp41 Trimers Containing the Fusion Peptide and Transmembrane Domains.

Human immunodeficiency virus (HIV) is membrane-enveloped, and an initial infection step is joining/fusion of viral and cell membranes. This step is catalyzed by gp41, which is a single-pass integral viral membrane protein. The protein contains an ∼170-residue ectodomain located outside the virus that is important for fusion and includes the fusion peptide (FP), N-helix, loop, C-helix, and viral membrane-proximal external region (MPER). The virion initially has noncovalent complexes between three gp41 ectodomains and three gp120 proteins. A gp120 contains ∼500 residues and functions to identify target T-cells and macrophages via binding to specific protein receptors of the target cell membrane. gp120 moves away from the gp41 ectodomain, and the ectodomain is thought to bind to the target cell membrane and mediate membrane fusion. The secondary and tertiary structures of the ectodomain are different in the initial complex with gp120 and the final state without gp120. There is not yet imaging of gp41 during fusion, so the temporal relationship between the gp41 and membrane structures is not known. This study describes biophysical and functional characterization of large gp41 constructs that include the ectodomain and transmembrane domain (TM). Significant fusion is observed of both neutral and anionic vesicles at neutral pH, which reflects the expected conditions of HIV/cell fusion. Fusion is enhanced by the FP, which in HIV/cell fusion likely contacts the host membrane, and the MPER and TM, which respectively interfacially contact and traverse the HIV membrane. Initial contact with vesicles is made by protein trimers that are in a native oligomeric state that reflects the initial complex with gp120 and also is commonly observed for the ectodomain without gp120. Circular dichroism data support helical structure for the N-helix, C-helix, and MPER and nonhelical structure for the FP and loop. Distributions of monomer, trimer, and hexamer states are observed by size-exclusion chromatography (SEC), with dependences on solubilizing detergent and construct. These SEC and other data are integrated into a refined working model of HIV/cell fusion that includes dissociation of the ectodomain into gp41 monomers followed by folding into hairpins that appose the two membranes, and subsequent fusion catalysis by trimers and hexamers of hairpins. The monomer and oligomer gp41 states may therefore satisfy dual requirements for HIV entry of membrane apposition and fusion.

Influenza Hemagglutinin Fusion Domain by Advanced NMR using Novel Orthagonal Refinement, BICS Curvature Measurements and Native Lipid Environments

The influenza fusion peptide domain (HAfp23) is comprised of the N‐terminal 23 residues of the hemagglutinin glycoprotein which are highly conserved and have been shown to be essential for membrane fusion and the infection process of the Influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We reveal that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.Our investigation of the HAfp23 system led to our development of novel NMR techniques including Bicelle Induced Curvature and Sorting (BICS) and application of a new positively charged alignment media (Pinacyanol Acetate) for structure refinement. These methods were applied to the study of HAfp23 in combination with well‐established NMR techniques, differential scanning calorimetry (DSC) and fluorescent lipid mixing assays. Furthermore, we have developed an NMR based approach to study HAfp23 in a more native lipid environment which we anticipate to be highly useful in the study of membrane proteins. The elucidations made on HAfp23 as well as the application of novel techniques to study membrane proteins are of high interest to the biophysical communities.Support or Funding InformationThis work was supported by the Department of Chemistry at the University of Illinois at Chicago (UIC) and a UIC Campus Research Board grant. Portions of this work were carried out in the Center for Structural Biology via the Research Resources Center at UIC.

A computational model for predicting transmembrane regions of retroviruses.

Transmembrane region (TR) is a conserved region of transmembrane (TM) subunit in envelope (env) glycoprotein of retrovirus. Evidences have shown that TR is responsible for anchoring the env glycoprotein on the lipid bilayer and substitution of the TR for a covalently linked lipid anchor abrogates fusion. However, universal software could not achieve sufficient accuracy as TM in env also has several motifs such as signal peptide, fusion peptide and immunosuppressive domain composed largely of hydrophobic residues. In this paper, a support vector machine-based (SVM) model is proposed to identify TRs in retroviruses. Firstly, physicochemical and evolutionary information properties were extracted as original features. And then, the feature importance was analyzed by minimum Redundancy Maximum Relevance (mRMR) feature selection criterion. Our model achieved an Sn of 0.955, Sp of 0.998, ACC of 0.995, MCC of 0.954 using 10-fold cross-validation on the training dataset. These results suggest that the proposed model can be used to predict TRs in non-annotation retroviruses and 11917, 3344, 2, 289 and 6 new putative TRs were found in HERV, HIV, HTLV, SIV, MLV, respectively.

Solid-State Nuclear Magnetic Resonance Investigation of the Structural Topology and Lipid Interactions of a Viral Fusion Protein Chimera Containing the Fusion Peptide and Transmembrane Domain.

The fusion peptide (FP) and transmembrane domain (TMD) of viral fusion proteins play important roles during virus-cell membrane fusion, by inducing membrane curvature and transient dehydration. The structure of the water-soluble ectodomain of viral fusion proteins has been extensively studied crystallographically, but the structures of the FP and TMD bound to phospholipid membranes are not well understood. We recently investigated the conformations and lipid interactions of the separate FP and TMD peptides of parainfluenza virus 5 (PIV5) fusion protein F using solid-state nuclear magnetic resonance. These studies provide structural information about the two domains when they are spatially well separated in the fusion process. To investigate how these two domains are structured relative to each other in the postfusion state, when the ectodomain forms a six-helix bundle that is thought to force the FP and TMD together in the membrane, we have now expressed and purified a chimera of the FP and TMD, connected by a Gly-Lys linker, and measured the chemical shifts and interdomain contacts of the protein in several lipid membranes. The FP-TMD chimera exhibits α-helical chemical shifts in all the membranes examined and does not cause strong curvature of lamellar membranes or membranes with negative spontaneous curvature. These properties differ qualitatively from those of the separate peptides, indicating that the FP and TMD interact with each other in the lipid membrane. However, no 13C-13C cross peaks are observed in two-dimensional correlation spectra, suggesting that the two helices are not tightly associated. These results suggest that the ectodomain six-helix bundle does not propagate into the membrane to the two hydrophobic termini. However, the loosely associated FP and TMD helices are found to generate significant negative Gaussian curvature to membranes that possess spontaneous positive curvature, consistent with the notion that the FP-TMD assembly may facilitate the transition of the membrane from hemifusion intermediates to the fusion pore.

Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection

Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4+ lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

A novel bispecific peptide HIV-1 fusion inhibitor targeting the N-terminal heptad repeat and fusion peptide domains in gp41.

HIV-1 fusion with the target cell is initiated by the insertion of the gp41 fusion peptide (FP) into the target cell membrane and the interaction between the gp41N- and C-terminal heptad repeats (NHR and CHR), followed by the formation of the six-helix bundle (6-HB) fusion core. Therefore, both FP and NHR are important targets for HIV-1 fusion inhibitors. Here, we designed and synthesized a dual-target peptidic HIV-1 fusion inhibitor, 4HR-LBD-VIRIP, in which 4HR-LBD is able to bind to the gp41 NHR domain, while VIRIP is able to interact with gp41 FP. We found that 4HR-LBD-VIRIP is about tenfold more potent than 4HR-LBD and VIRIP in inhibiting HIV-1IIIB infection and HIV-1 envelope glycoprotein (Env)-mediated cell-cell fusion, suggesting that this dual-target HIV-1 fusion inhibitor possesses a strong synergistic antiviral effect. A biophysical analysis indicates that 4HR-LBD-VIRIP can interact with N70 peptide that contains the gp41 NHR and FP domains and binds with lipid membrane. This study provides a new approach for designing novel viral fusion inhibitors against HIV and other enveloped viruses with class I membrane fusion proteins.

Structure and Mechanisms of Actions of Curvature-Inducing Viral Membrane Proteins from Solid-State NMR

Many membrane proteins such as viral fusion proteins induce membrane curvature for function. To understand how protein structure and membrane properties underlie such curvature induction, we have applied and developed solid-state NMR spectroscopic techniques. The conformation, topology, and lipid interactions of the fusion peptide (FP) and transmembrane domain (TMD) of a paramyxovirus fusion protein are investigated. 13C and 15N chemical shifts indicate that both the FP and TMD adopt lipid-dependent conformations, with the α-helical conformation dominant in low-curvature phosphocholine and phosphoglycerol membranes while the β-strand conformation dominant in negative-curvature phosphoethanolamine (PE) membranes. 31P and 2H NMR spectra, together with small-angle X-ray scattering results, indicate that the PE membrane is transformed by the TMD into a bicontinuous cubic phase, which is rich in negative Gaussian curvature, the type of curvature in hemifusion intermediates and fusion pores. These results strongly suggest that the two hydrophobic termini of this viral fusion protein actively facilitate membrane topological changes to promote membrane merger, and the β-strand is the fusogenic conformation.To detect membrane curvature and the binding site of proteins in mixed-curvature phospholipid membranes, we have developed a solid-state NMR approach based on magnetically oriented bicelles, relaxation NMR and 2D correlation experiments under off-magic-angle spinning (OMAS). We demonstrate this approach on the influenza M2 protein, which mediates virus budding by membrane scission. An M2 peptide encompassing the TMD and an amphipathic helix in the cytoplasmic domain is compared with the TMD alone. Our results indicate that the amphipathic helix causes high membrane curvature, moreover, the peptide preferentially partitions into this high-curvature domain. This bicelle-based solid-state NMR approach is generally applicable to curvature-inducing membrane proteins.

Membrane Fusion and Infection of the Influenza Hemagglutinin.

The influenza virus is a major health concern associated with an estimated 5000 to 30,000 deaths every year (Reed et al. 2015) and a significant economic impact with the development of treatments, vaccinations and research (Molinari et al. 2007). The entirety of the influenza genome is comprised of only eleven coding genes. An enormous degree of variation in non-conserved regions leads to significant challenges in the development of inclusive inhibitors for treatment. The fusion peptide domain of the influenza A hemagglutinin (HA) is a promising candidate for treatment since it is one of the most highly conserved sequences in the influenza genome (Heiny et al. 2007), and it is vital to the viral life cycle. Hemagglutinin is a class I viral fusion protein that catalyzes the membrane fusion process during cellular entry and infection. Impediment of the hemagglutinin's function, either through incomplete post-translational processing (Klenk et al. 1975; Lazarowitz and Choppin 1975) or through mutations (Cross et al. 2001), leads to non-infective virus particles. This review will investigate current research on the role of hemagglutinin in the virus life cycle, its structural biology and mechanism as well as the central role of the hemagglutinin fusion peptide (HAfp) to influenza membrane fusion and infection.