A highly membrane-active peptide in Flock House virus: implications for the mechanism of nodavirus infection.

BACKGROUND: Nodaviruses are among the simplest animal viruses, and are therefore attractive systems for deconvoluting core viral processes such as assembly, infection and uncoating. Membrane translocation of the single-stranded RNA genome of nodaviruses has been proposed to be mediated by direct lipid-protein interactions between a post-assembly autocatalytic cleavage product from the capsomere and the target membrane. To probe the validity of this hypothesis, we have synthesized a 21-residue Met-->Nle (norleucine) variant of the amino-terminal helical domain (denoted here as gamma1) of the cleavage peptide in Flock House nodavirus (FHV) and studied its ability to alter membrane structure and function. RESULTS: The synthetic peptide gamma1 increases membrane permeability to hydrophilic solutes, as judged by fluorescence experiments with liposome-encapsulated dyes and ion-conductance measurements. Furthermore, peptide orientation and location within lipid bilayers was determined using tryptophan-fluorescence-quenching experiments and attenuated total reflectance infrared spectroscopy. CONCLUSIONS: The helical domain of the FHV cleavage product partitions spontaneously into lipid bilayers and increases membrane permeability, consistent with the postulated mechanism for viral genome translocation. The existence of a membrane-binding domain in the FHV cleavage sequence suggests peptide-triggered disruption of the endosomal membrane as a prelude to viral uncoating in the host cytoplasm. A model for this interaction is proposed.     

Targeting cell entry of enveloped viruses as an antiviral strategy

The entry of enveloped viruses into their host cells involves several successive steps, each one being amenable to therapeutic intervention. Entry inhibitors act by targeting viral and/or cellular components, through either the inhibition of protein-protein interactions within the viral envelope proteins or between viral proteins and host cell receptors, or through the inhibition of protein-lipid interactions. Interestingly, inhibitors that concentrate into/onto the membrane in order to target a protein involved in the entry process, such as arbidol or peptide inhibitors of the human immunodeficiency virus (HIV), could allow the use of doses compatible with therapeutic requirements. The efficacy of these drugs validates entry as a point of intervention in viral life cycles. Strategies based upon small molecule antiviral agents, peptides, proteins or nucleic acids, would most likely prove efficient in multidrug combinations, in order to inhibit several steps of virus life cycle and prevent disease progression.     

13C-13C and (15)N-(13)C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: amino acid-type assignments and evidence for multiple conformations

Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an approximately 20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform (13)C, (15)N labeling over large contiguous regions of amino acids. Two-dimensional (13)C-(13)C and (15)N-(13)C spectra were obtained for the membrane-bound fusion peptides and an amino acid-type (13)C assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the (13)C chemical shifts were more consistent with beta strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with beta strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous (13)C and (15)N assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Calpha nuclei. These sets of two shifts may indicate two beta strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4 T instrument, one (13)C-(13)C correlation spectrum was obtained on a 16.4 T instrument and was better resolved than the comparable 9.4 T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.     

Nanoassembly of Oligopeptides and DNA Mimics the Sequential Disassembly of a Spherical Virus

Protein subunits of a low aspect ratio (width over length) with stimuli‐responsiveness are hallmark building blocks of spherical viruses. The interaction of these repeating subunits enables hierarchical assembly for genome packaging and sequential disassembly for optimal genome release. Here, we mimicked these features and constructed a functional spherical artificial virus. The rationally designed 22‐amino acid peptide containing pH‐sensitive histidines and aromatic residues self‐assembled into homogenous nanodiscs of a low aspect ratio (≈7×7×4 nm). In the presence of DNA, the inter‐nanodisc interactions drove the formation of a viral capsid‐like structure enclosing DNA in the interior. This artificial virus roughly 50 nm in diameter underwent partial disassembly in response to acidic pH. The resulting intermediate with lowered DNA‐binding affinity continued to protect DNA from nuclease digestion. Such nanostructures, which mimic the intricate morphology and the intracellular transformation of spherical viruses, can be useful for constructing synthetic gene delivery vehicles, as evidenced by their efficient transgene expression.     

Synthesis and membrane binding properties of a lipopeptide fragment from influenza virus a hemagglutinin

Hemagglutinin from influenza virus A is a S-palmitoylated lipoglycoprotein in which the lipid groups are thought to influence the interaction between cell membrane and capsid during budding of viral offspring as well as fusion processes of the viral membrane with the endosome after entry of the viral particle into the cell. The paper describes the development of a method for the synthesis of characteristic lipidated hemagglutinin derived peptides which additionally carry the fluorescent 7-nitrobenz-2oxa-1,3-diazole (NBD) group. To achieve this goal the enzyme-sensitive para-phenylacetoxybenzyloxycarbonyl (PAOB) ester was developed. It is cleaved from the peptides and lipidated peptides under very mild conditions and with complete selectivity by treatment with the enzyme penicillin G acylase; this results in the formation of a phenolate. This intermediate spontaneously undergoes fragmentation thereby releasing the desired carboxylates. The combined use of this enzyme-labile fragmenting ester with the acid-labile Boc group, the Pd(0)-sensitive allyl ester and the corresponding Aloc urethane gave access to a mono-S-palmitoylated and a doubly S-palmitoylated NBD-labelled hemagglutinin peptide. The binding of these lipopeptides to model membranes was analyzed in a biophysical setup monitoring the transfer of fluorescent-labelled lipopeptide from vesicles containing the non-exchangeable fluorescence quencher Rho-DHPE to quencher-free vesicles. The experiments demonstrate that one lipid group is not sufficient for quasi-irreversible membrane insertion of lipidated peptides. This is, however, achieved by introduction of the bis-palmitoyl anchor. The intervesicle transfer always implies release of peptides localized at the outer face of the vesicles into solution followed by diffusion to and insertion into acceptor vesicles. For peptides bound at the inner face of the vesicle membrane, however, an additional flip-flop diffusion to the outer face has to occur beforehand. The kinetics of these processes were estimated by fast chemical quench of the outside fluorophores by sodium dithionite.     

Mechanical characterization of HIV‐1 with a solid‐state nanopore sensor

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo‐type human immunodeficiency virus type 1 at sub‐micron scale. Non‐infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.     

An Intrinsically Disordered Peptide Facilitates Non‐Endosomal Cell Entry

Many cell‐penetrating peptides (CPPs) fold at cell surfaces, adopting α‐ or β‐structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane‐lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non‐endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum‐stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane‐impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.     

Peptide inhibitors of viral assembly: a novel route to broad-spectrum antivirals

We investigated the potential of small peptide segments to function as broad-spectrum antiviral drug leads. We extracted the α-helical peptide segments that share common secondary-structure environments in the capsid protein-protein interfaces of three unrelated virus classes (PRD1-like, HK97-like, and BTV-like) that encompass different levels of pathogenicity to humans, animals, and plants. The potential for the binding of these peptides to the individual capsid proteins was then investigated using blind docking simulations. Most of the extracted α-helical peptides were found to interact favorably with one or more of the protein-protein interfaces within the capsid in all three classes of virus. Moreover, binding of these peptides to the interface regions was found to block one or more of the putative "hot spot" regions on the protein interface, thereby providing the potential to disrupt virus capsid assembly via competitive interaction with other capsid proteins. In particular, binding of the GDFNALSN peptide was found to block interface "hot spot" regions in most of the viruses, providing a potential lead for broad-spectrum antiviral drug therapy.     

Cell Surface Assembly of HIV gp41 Six-Helix Bundles for Facile, Quantitative Measurements of Hetero-oligomeric Interactions

Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils.     

pH-induced destabilization of lipid bilayers by a peptide from the VP3 protein of the capsid of hepatitis A virus.

The membrane destabilizing and fusogenic properties of the synthetic peptide VP3(110-121), corresponding to an immunogenic sequence of the hepatitis A virus (HAV) VP3 capsid protein, were studied. By tryptophan fluorescence and acryalmide quenching it was demonstrated that the peptide binds liposomes of POPC-SM-DPPE (47 + 39 + 14) and POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) and penetrates the membrane, at both neutral and acidic pH (POPC = 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM = sphingomyelin; DPPE = 1,2-dipalmitoylphosphatidylethanolamine; DOTAP = 1,2-dioleoyl-3-trimethylammoniumpropane). VP3(110-121) did not have membrane-destabilizing properties at neutral pH. Acid-induced destabilization of the vesicles was demonstrated by fluorescence techniques and dynamic light scattering. VP3(110-121) induced aggregation of POPC-SM-DPPE-DOTAP (40 + 33 + 12 + 15) vesicles, lipid mixing and leakage of vesicle contents, all consistent with fusion of vesicles. In POPC-SM-DPPE (47 + 39 + 14) vesicles, at acidic pH, VP3(110-121) induced membrane destabilization with leakage of contents but without aggregation of vesicles or lipid mixing. The peptide only showed fusogenic properties when bound to the vesicles at neutral pH before acidification to pH below 6.0, and no effect was seen if the peptide was added to vesicles already set at acidic pH. These results may have physiological significance in the mechanism of infection of host hepatic cells by HAV.     

Structural variety of membrane permeable peptides

Peptide-mediated protein delivery into living cells has been attracting our attention. Among the peptides that have been reported to have carrier activity, the one from the human immunodeficient virus (HIV)-1 Tat has been most often used for the introduction of exogenous macromolecules into cells. We have shown that not only the Tat peptide, but also various arginine-rich peptides showed very similar characteristics in translocation, and the possible presence of ubiquitous internalization mechanisms among the arginine-rich peptides has also been suggested. These arginine-rich peptides includes ones derived from HIV-1 Rev and flock house virus coat proteins. The linear- and branched-chain peptides containing approximately 8 residues of arginine also show a similar ability. In this review, we present the structural variety of membrane permeable peptides and provide a survey of the findings on the translocation of these peptides through the cell membranes.     

Synthesis,structural characterization,and immunological properties of carbon nanotubes functionalized with peptides

Carbon nanotubes (NTs) are becoming highly attractive molecules for applications in medicinal chemistry. The main problem of insolubility in aqueous media has been solved by developing a synthetic protocol that allows highly water-soluble carbon NTs to be obtained. As a result, biologically active peptides can be easily linked through a stable covalent bond to carbon NTs. We have demonstrated that a bound peptide from the foot-and-mouth disease virus, corresponding to the 141-159 region of the viral envelope protein VP1, retained the structural integrity and was recognized by monoclonal and polyclonal antibodies. In addition, this peptide-NT conjugate is immunogenic, eliciting antibody responses of the right specificity. Such a system could be greatly advantageous for diagnostic purposes and could find future applications in vaccine delivery.     

Cellular uptake of an α-AApeptide

Some short and cationic peptides such as the Tat peptide can cross the cell membrane and function as vectors for intracellular delivery. Here we show that an α-AApeptide is able to penetrate the membranes of living cells from an extracellular environment and enter the endosome and cytoplasm of cells. The efficiency of the cellular uptake is comparable to a Tat peptide (48-57) of the same length and is unexpectedly superior to an α-peptide with identical functional groups. The mechanism of uptake is similar to that of the Tat peptide and is through endocytosis by an energy-dependent pathway. Due to the easy synthesis of the α-AApeptides, their resistance to proteolytic hydrolysis, and their low cytotoxicity, α-AApeptides represent a new class of transporters for the delivery of drugs.     

Phototoxicity against tumor cells and Sindbis virus by an octahedral rhodium bisbipyridyl complex and evidence for the genome as a target in viral photoinactivation

An octahedral rhodium complex (cis-dichloro(dipyrido[3,2a-2',3'c]phenazine)(1,10-phenanthroline)rhodium(III) chloride; DPPZPHEN) has been prepared that can penetrate tumor cell membranes and the Sindbis viral capsid. The compound is phototoxic to these entities when irradiated with UVA light. Model studies with calf thymus and supercoiled plasmid DNA indicate that the complex can both bind with, and nick, nucleic acid. Analysis of Sindbis virus, following irradiation with the metal complex, confirmed that the viral genome was rendered noninfectious by this treatment.     

单病毒示踪

病毒是对人类健康威胁最大的病原之一,由其引发的病毒性疾病对人民健康、国家安全和社会经济构成重大威胁.病毒感染机制研究对病毒性疾病的防控及治疗具有重大意义.病毒侵染宿主细胞的动态过程涉及病毒组分与多种细胞组分或细胞器间复杂的相互作用,但是传统手段无法对该动态过程进行实时跟踪研究.单病毒示踪技术作为一种可以实时原位示踪单颗...     

DIFFERENTIAL EFFECTS OF PHOTOACTIVE FURANYL COMPOUNDS ON VIRUS FUNCTIONS

Abstract— Five photoactive furanyl compounds were investigated for their activities against viruses. The two furanocoumarins used were 8-methoxypsoralen (8-MOP) and angelicin; two furanochromones, visnagin and khellin, and the furanoquinoline, dictamnine, were also used. The DNA-containing herpes virus murine cytomegalovirus (MCMV) and the RNA-containing togavirus, Sindbis virus, were the target viruses. All five compounds inactivated both viruses in the presence of UVA, although Sindbis virus was much less sensitive. The relative order of antiviral potency was 8-MOP > dictamnine > visnagin > angelicin > khellin. Dictamnine however was slightly more effective than 8-MOP against Sindbis virus. None of the treatments affected the structural integrity of MCMV, nor did they interfere with the normal transit of the virus into host cells or the localisation of the viral genome in the cell nucleus. Some early viral gene functions were expressed but the viruses did not replicate.     

Interaction of a peptide from the pre-transmembrane domain of the severe acute respiratory syndrome coronavirus spike protein with phospholipid membranes

The severe acute respiratory syndrome coronavirus (SARS-CoV) envelope spike (S) glycoprotein, a Class I viral fusion protein, is responsible for the fusion between the membranes of the virus and the target cell. In order to gain new insight into the protein membrane alteration leading to the viral fusion mechanism, a peptide pertaining to the putative pre-transmembrane domain (PTM) of the S glycoprotein has been studied by infrared and fluorescence spectroscopies regarding its structure, its ability to induce membrane leakage, aggregation, and fusion, as well as its affinity toward specific phospholipids. We demonstrate that the SARS-CoV PTM peptide binds to and interacts with phospholipid model membranes, and, at the same time, it adopts different conformations when bound to membranes of different compositions. As it has been already suggested for other viral fusion proteins such as HIV gp41, the region of the SARS-CoV protein where the PTM peptide resides could be involved in the merging of the viral and target cell membranes working synergistically with other membrane-active regions of the SARS-CoV S glycoprotein to heighten the fusion process and therefore might be essential for the assistance and enhancement of the viral and cell fusion process.     

Approaches to the Potential Therapy of COVID-19: A General Overview from the Medicinal Chemistry Perspective

In spite of advances in vaccination, control of the COVID-19 pandemic will require the use of pharmacological treatments against SARS-CoV2. Their development needs to consider the existence of two phases in the disease, namely the viral infection and the inflammatory stages. The main targets for antiviral therapeutic intervention are: (a) viral proteins, including the spike (S) protein characteristic of the viral cover and the viral proteases in charge of processing the polyprotein arising from viral genome translation; (b) host proteins, such as those involved in the processes related to viral entry into the host cell and the release of the viral genome inside the cell, the elongation factor eEF1A and importins. The use of antivirals targeted at host proteins is less developed but it has the potential advantage of not being affected by mutations in the genome of the virus and therefore being active against all its variants. Regarding drugs that address the hyperinflammatory phase of the disease triggered by the so-called cytokine storm, the following strategies are particularly relevant: (a) drugs targeting JAK kinases; (b) sphingosine kinase 2 inhibitors; (c) antibodies against interleukin 6 or its receptor; (d) use of the traditional anti-inflammatory corticosteroids.     

Construction and activity of a synthetic basement membrane with active laminin peptides and polysaccharides

Cell-adhesive peptides derived from extracellular matrix (ECM) proteins are potential candidates for incorporating cell-binding activities into materials for tissue engineering. We have identified a number of cell adhesive peptides from laminins, which are major components of basement membrane ECM. Our goal is the development of synthetic basement membranes using the peptides on scaffolds. We review peptide–polysaccharide complexes, which were prepared by conjugation of the peptides to chitosan and alginate, and the biological activities of the resulting matrices. The peptide–polysaccharide matrices can also be used as a biomaterial for cell transplantation. These studies suggest that the peptide–polysaccharide complexes have the potential to mimic the multifunctional basement membrane and may be useful for tissue engineering.     

Molecular Docking of SP40 Peptide towards Cellular Receptors for Enterovirus 71 (EV-A71)

Enterovirus 71 (EV-A71) is one of the predominant etiological agents of hand, foot and mouth disease (HMFD), which can cause severe central nervous system infections in young children. There is no clinically approved vaccine or antiviral agent against HFMD. The SP40 peptide, derived from the VP1 capsid of EV-A71, was reported to be a promising antiviral peptide that targeted the host receptor(s) involved in viral attachment or entry. So far, the mechanism of action of SP40 peptide is unknown. In this study, interactions between ten reported cell receptors of EV-A71 and the antiviral SP40 peptide were evaluated through molecular docking simulations, followed by in vitro receptor blocking with specific antibodies. The preferable binding region of each receptor to SP40 was predicted by global docking using HPEPDOCK and the cell receptor-SP40 peptide complexes were refined using FlexPepDock. Local molecular docking using GOLD (Genetic Optimization for Ligand Docking) showed that the SP40 peptide had the highest binding score to nucleolin followed by annexin A2, SCARB2 and human tryptophanyl-tRNA synthetase. The average GoldScore for 5 top-scoring models of human cyclophilin, fibronectin, human galectin, DC-SIGN and vimentin were almost similar. Analysis of the nucleolin-SP40 peptide complex showed that SP40 peptide binds to the RNA binding domains (RBDs) of nucleolin. Furthermore, receptor blocking by specific monoclonal antibody was performed for seven cell receptors of EV-A71 and the results showed that the blocking of nucleolin by anti-nucleolin alone conferred a 93% reduction in viral infectivity. Maximum viral inhibition (99.5%) occurred when SCARB2 was concurrently blocked with anti-SCARB2 and the SP40 peptide. This is the first report to reveal the mechanism of action of SP40 peptide in silico through molecular docking analysis. This study provides information on the possible binding site of SP40 peptide to EV-A71 cellular receptors. Such information could be useful to further validate the interaction of the SP40 peptide with nucleolin by site-directed mutagenesis of the nucleolin binding site.