Surface Display of Rice Stripe Virus NSvc2 and Analysis of Its Membrane Fusion Activity

Rice stripe virus (RSV) infects rice and is transmitted in a propagative manner by the small brown planthopper. How RSV enters an insect cell to initiate the infection cycle is poorly understood. Sequence analysis revealed that the RSV NSvc2 protein was similar to the membrane glycoproteins of several members in the family Bunyaviridae and might induce cell membrane fusion. To conveniently study the membrane fusion activity of NSvc2, we constructed cell surface display vectors for expressing Nsvc2 on the insect cell surface as the membrane glycoproteins of the enveloped viruses. Our results showed that NSvc2 was successfully expressed and displayed on the surface of insect Sf9 cells. When induced by low pH, the membrane fusion was not observed in the cells that expressed NSvc2. Additionally, the membrane fusion was also not detected when co-expressing Nsvc2 and the viral capsid protein on insect cell surface. Thus, RSV NSvc2 is probably different from the phlebovirus counterparts, which could suggest different functions. RSV might enter insect cells other than by fusion with plasma or endosome membrane.

Simulation and prediction of membrane fusion dynamics

Membrane fusion is an important process by which biological membranes perform their life activities. Simulations show that the membrane fusion process happens mainly through three pathways, where the Stalk-Pore hypothesis, in which two membranes come into close contact to form a stalk to a hemifusion intermediate, and then the fusion pore opens to achieve completely fusion, is widely accepted, and there exist two free energy barriers that break the current structural steady state for lipid rearrangement. Factors of lipid composition, mechanical environment, protein and ion have regulatory roles in the membrane fusion process by effecting membrane curvature structurally and the free energy barriers from energetic perspective. Meanwhile, many theoretical models, represented by the Helfrich model, have been proposed to predict the membrane fusion process. In this paper, we review the research process of membrane fusion and mainly introduce the dynamics of membrane fusion, regulation factors and typical theoretical models.

Sum Frequency Spectroscopy Studies on Cell Membrane Fusion Induced by Divalent Cations

Cell membrane fusion is a fundamental biological process involved in a number of cellular living functions. Regarding this, divalent cations can induce fusion of the lipid bilayers through binding and bridging of divalent cations to the charged lipids, thus leading to the cell membrane fusion. However, the elaborate mechanism of cell membrane fusion induced by divalent cations is still needed to be elucidated.Here, surface/interface sensitive sum frequency generation vibrational spectroscopy(SFG-VS) and dynamic light scattering(DLS) were applied in this research to study the responses of phospholipid monolayer to the exposure of divalent metal ions i.e.Ca^(2+)and Mg^(2+). According to the particle size distribution results measured by DLS experiments, it was found that Ca^(2+)could induce inter-vesicular fusion while Mg^(2+)could not. An octadecyltrichlorosilane self-assembled monolayer(OTS SAM)-lipid monolayer system was designed to model the cell membrane for the SFG-VS experiment. Ca^(2+)could interact with the lipid POO_(2)^(-)head groups more strongly, resulting in cell membrane fusion more easily, in comparison with Mg^(2+). No specific interaction between the two metal cations and the C=O groups was observed. However, the C=O orientations changed more after Ca^(2+)-PO2-binding than Mg^(2+)mediation on lipid monolayer. Meanwhile, Ca^(2+)could induce dehydration of the lipids(which should be related to the strong Ca^(2+)-PO_(2)^(-)interaction), leading to the reduced hindrance for cell membrane fusion.

Deletion of the first glycosylation site promotes Lassa virus glycoprotein-mediated membrane fusion

The Lassa virus(LASV)is endemic in West Africa and causes severe hemorrhagic Lassa fever in humans.The glycoprotein complex(GPC)of LASV is highly glycosylation-modified,with 11 N-glycosylation sites.All 11 N-linked glycan chains play critical roles in GPC cleavage,folding,receptor binding,membrane fusion,and immune evasion.In this study,we focused on the first glycosylation site because its deletion mutant(N79Q)results in an unexpected enhanced membrane fusion,whereas it exerts little effect on GPC expression,cleavage,and receptor binding.Meanwhile,the pseudotype virus bearing GPC_(N79Q)was more sensitive to the neutralizing antibody 37.7H and was attenuated in virulence.Exploring the biological functions of the key glycosylation site on LASV GPC will help elucidate the mechanism of LASV infection and provide strategies for the development of attenuated vaccines against LASV infection.

Membrane fusion reverse micelle platforms as potential oral nanocarriers for efficient internalization of free hydrophilic peptides

Orally administered peptides or proteins are garnering increasing preference owing to their superiority in terms of patient compliance and convenience.However,the development of oral protein formulations has stalled due to the low bioavailability of macromolecules that encounter the aggressive gastrointestinal environment and harsh mucus villi barrier.Herein,we propose an ideal reverse micelle/self-emulsifying drug delivery system(RM/SEDDS)nanoplatform that is capable of improving the oral bioavailability of hydrophilic peptides by preventing enzymatic degradation and enhancing mucosal permeability.Upon the passage through the mucus,the self-emulsifying drug delivery system with optimal surface properties effectively penetrates the viscoelastic mucosal barrier,followed by the exposure of the inner reverse micelle amphipathic vectors,which autonomously form continua with the lipidic cell membrane and facilitate the internalization of drugs.This membrane-fusion mechanism inaugurates a new way for hydrophilic peptide delivery in the free form,circumventing the traditional impediments of the cellular internalization of nanocarriers and subsequent poor release of drugs.And more importantly,reverse micelles are not spatially specific to the laden drugs,which enables their delivery for a myriad of peptide clinical drugs.In conclusion,as an exquisitely designed nanoplatform,RM/SEDDS overcomes multiple physiological barriers and opens a new path for drug cellular entry,providing new prospects for the development of oral drug delivery systems.

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion

Despite the high mutation rate of HIV-1,the amino acid sequences of the membrane-spanning domain(MSD)of HIV-1 gp41 are well conserved.Arginine residues are rarely found in single membrane-spanning domains,yet an arginine residue,R696(the numbering is based on that of HXB2),is highly conserved in HIV-1 gp41.To examine the role of R696,it was mutated to K,A,I,L,D,E,N,and Q.Most of these substitutions did not affect the expression,processing or surface distribution of the envelope protein(Env).However,a syncytia formation assay showed that the substitution of R696 with amino acid residues other than K,a naturally observed mutation in the gp41 MSD,decreased fusion activity.Substitution with hydrophobic amino acid residues(A,I,and L)resulted in a modest decrease,while substitution with D or E,potentially negatively-charged residues,almost abolished the syncytia formation.All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein(DSP)assay that scores the degree of membrane fusion based on pore formation between fusing cells.Interestingly,the D and E substitutions did show some fusion activity in the DSP assays,suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability.However,nascent pores failed to develop,due probably to impaired activity in the pore enlargement process.Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Enhanced sampling for lipid-protein interactions during membrane dynamics

The inflexible concept of membrane curvature as an independent property of lipid structures is today obsolete.Lipid bilayers behave as many-body entities with emergent properties that depend on their interactions with the environment.In particular,proteins exert crucial actions on lipid molecules that ultimately condition the collective properties of the membranes.In this review,the potential of enhanced molecular dynamics to address cell-biology problems is discussed.The cases of membrane deformation,membrane fusion,and the fusion pore are analyzed from the perspective of the dimensionality reduction by collective variables.Coupled lipid-protein interactions as fundamental determinants of large membrane remodeling events are also commented.Finally,novel strategies merging cell biology and physics are considered as future lines of research.

M6PR interacts with the HA2 subunit of influenza A virus to facilitate the fusion of viral and endosomal membranes

Influenza A virus(IAV) commandeers numerous host cellular factors for successful replication. However, very few host factors have been revealed to be involved in the fusion of viral envelope and late endosomal membranes. In this study, we identified cation-dependent mannose-6-phosphate receptor(M6PR) as a crucial host factor for the replication of IAV. We found that siRNA knockdown of M6PR expression significantly reduced the growth titers of different subtypes of IAV, and that the inhibitory effect of M6PR siRNA treatment on IAV growth was overcome by the complement of exogenously expressed M6PR. When A549 cells were treated with siRNA targeting M6PR,the nuclear accumulation of viral nucleoprotein(NP) was dramatically inhibited at early timepoints post-infection, indicating that M6PR engages in the early stage of the IAV replication cycle. By investigating the role of M6PR in the individual entry and post-entry steps of IAV replication, we found that the downregulation of M6PR expression had no effect on attachment, internalization, early endosome trafficking,or late endosome acidification. However, we found that M6PR expression was critical for the fusion of viral envelope and late endosomal membranes. Of note, M6PR interacted with the hemagglutinin(HA) protein of IAV, and further studies showed that the lumenal domain of M6PR and the ectodomain of HA2 mediated the interaction and directly promoted the fusion of the viral and late endosomal membranes,thereby facilitating IAV replication. Together, our findings highlight the importance of the M6PR–HA interaction in the fusion of viral and late endosomal membranes during IAV replication.

Modeling biomembranes and red blood cells by coarse-grained particle methods

In this work, the previously developed coarse-grained （CG） particle models for biomembranes and red blood cells （RBCs） are reviewed, and the advantages of the CG particle methods over the continuum and atomistic simulations for modeling biological phenomena are discussed. CG particle models can largely increase the length scale and time scale of atomistic simulations by eliminating the fast degrees of freedom while preserving the mesoscopic structures and properties of the simulated system. Moreover, CG particle models can be used to capture the microstructural alternations in diseased RBCs and simulate the topological changes of biomembranes and RBCs, which are the major challenges to the typical continuum representations of membranes and RBCs. The power and versatility of CG particle methods are demonstrated：through simulating the dynamical processes mvolving significant topological .changes e.g. lipid self-assembly vesicle fusion and membrane budding.

Mechanics of water pore formation in lipid membrane under electric field

Transmembrane water pores are crucial for substance transport through cell membranes via membrane fusion, such as in neural communication. However, the molecular mechanism of water pore formation is not clear. In this study, we apply all-atom molecular dynamics and bias-exchange metadynamics simulations to study the process of water pore formation under an electric field. We show that water molecules can enter a membrane under an electric field and form a water pore of a few nanometers in diameter. These water molecules disturb the interactions between lipid head groups and the ordered arrangement of lipids. Following the movement of water molecules, the lipid head groups are rotated and driven into the hydrophobic region of the membrane. The reorientated lipid head groups inside the membrane form a hydrophilic surface of the water pore. This study reveals the atomic details of how an electric field influences the movement of water molecules and lipid head groups, resulting in water pore formation.

Recent progresses of exosome-liposome fusions in drug delivery

Exosomes are membrane-bound nanoscale extracellular vesicles,which produced by almost all organisms.Due to the excellent biocompatibility,long circulation time as well as low immunogenicity,exosomes as naturally-derived drug delivery carriers have experienced explosive growth over the past decades.However,issues such as insufficient loading efficiency,heterogeneous delivery efficiency,uncontrollable targeting ability,and low production limit their wide application.Recently,the emerging exosome-liposome fusion strategy has become a potential approach to solve such issues.Thus,this review mainly focuses on the currently developed exosome-liposome fusion strategy and their application in drug delivery as well as disease treatment.This review aims to shed light on the advantages of fusion strategy in drug delivery and provides a better understanding for more rational design.The current challenge and future perspective regarding their clinical translation and application will also be discussed.

Molecular aspects of mammalian fertilization

Mammalian fertilization is a highly regulated process, much of which are not clearly understood. Here we presentsome information in order to elaborate a working hypothesis for this process, beginning with the sperm modifications inthe epidydimis up to sperm and egg plasmalemma interaction and fusion. We also discuss the still poorly understood ca-pacitation process, the phenomenon of sperm chemo-attraction that brings the capacitated sperm to interact with theoocyte vestments and certain aspects of the acrosome reaction. (Asian J Androl 2001 Dec; 3 : 243 - 249)

Coarse Grained Molecular Dynamics Simulation of Interaction between Hemagglutinin Fusion Peptides and Lipid Bilayer Membranes

Microscopic level interaction between fusion-peptides and lipid bilayer membranes plays a crucial role in membrane fusion,a key step of viral infection.In this paper,we use coarse-grained molecular dynamics(CGMD)simulations to study the interaction between hemagglutinin fusion-peptides and phospholipid bilayer membranes.With CGMD,we are able to simulate the interaction of fusion peptides with a relatively large piece of membrane for a sufficiently long time period,which is necessary for a detailed understanding of the fusion process.A conformation of the peptide with a kink at the level of phosphate group is obtained,consistent with NMR and EPR studies.Our results show that the N-terminal segment of the peptide inserts more deeply into the membrane bilayer compared to the C-terminal segment,as observed in previous experiments.Our simulations also show that the presence of fusion peptides inside the membrane may cause bilayer thinning and lipid molecule disorder.Finally,our results reveal that peptides tend to aggregate,indicating cluster formation as seen in many experiments.

A Protein Extracted from Mouse Sperm That Plays an Important Role in Fertilization

A membrane protein was isolated from mouse sperm heads that had undergone acrosomal reaction induced by C2+ ionophore, A 23187, which, with a molecular weight of 77.6 kd, shows capability to block egg-sperm fusion. As revealed by analysis usintg isotopic markers, this protein is one of the chief membrane proteins of inner acrosomal membrane or the outer membrane of equatorial segment and Post-acrosomal region; treatment of mouse sperms with 0.6 μg/ml of the Purified protein for 30 minutes reduced the sperm-egg fusion index by 51%.The above results led us to the conclusion that the protein is an active participant in sperm-egg fusion. The possible existence of sperm receptor on egg plasma membrane was discussed.

利用定量单病毒示踪技术探究SARS-CoV-2与细胞膜融合动态过程

Viral envelope fusion with the host plasma membrane(PM)for genome release is a hallmark step in the life cycle of many enveloped viruses.This process is regulated by a complex network of biomolecules on the PM,but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking.Here,we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events.This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)at the singlevirus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process.Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.

Identification of residues in Lassa virus glycoprotein 1 involved in receptor switch

Lassa virus(LASV)is an enveloped,negative-sense RNA virus that causes Lassa hemorrhagic fever.Successful entry of LASV requires the viral glycoprotein 1(GP1)to undergo a receptor switch from its primary receptor alpha-dystroglycan(α-DG)to its endosomal receptor lysosome-associated membrane protein 1(LAMP1).A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch.To test the hypothesis that other non-conserved residues also contribute to receptor switch,we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1.Four residues,L84,K88,L107,and H170,were identified as critical for receptor switch.Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus(LCMV)residue(L84 N,K88E,L10F,and H170S)reduced the binding affinity of LASV GP1 for LAMP1.Moreover,all mutations caused decreases in glycoprotein precursor(GPC)-mediated membrane fusion at both pH 4.5 and 5.2.The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types,while L107F and H170S had only mild effects on infectivity.Using biolayer light interferometry assay,we found that all four mutants had decreased binding affinity to LAMP1,in the order of binding affinity being L84 N>L107F>K88E>H170S.The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.

Clinical applications of exosome membrane proteins

Extracellular vesicles(EVs)are small membranous particles that can mediate cell-to-cell communication and which are divided into at least three categories according to their subcellular origin and size:exosomes,microvesicles,and apoptotic bodies.Exosomes are the smallest(30–150 nm)of these EVs,and play an important role in EV-mediated cell-to-cell interactions,by transferring proteins,nucleic acids and,lipids from their parental cells to adjacent or distant cells to alter their phenotypes.Most exosome studies in the past two decades have focused on their nucleic acid composition and their transfer ofmRNAs and microRNAs to neighboring cells.However,exosomes also carry specific membrane proteins that can identify the physiological and pathological states of their parental cells or indicate their preferential target cells or tissues.Exosome membrane protein expression can also be directly employed or modified to allow exosomes to serve as drug delivery systems and therapeutic platforms,including in targeted therapy approaches.This review will briefly summarize information on exosome membrane proteins components and their role in exosome–cell interactions,including proteins associated with specific cell-interactions and diseases,and the potential for using exosome membrane proteins in therapeutic targeting approaches.

Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Cholesterol represents one of the key constituents of small,dynamic,sterol-and sphingolipid-enriched domains on the plasma membrane.It has been reported that many viruses depend on plasma membrane cholesterol for efficient infection.In this study,the role of the plasma membrane cholesterol in porcine reproductive and respiratory syndrome virus(PRRSV) infection of MARC-145 cells was investigated.Pretreatment of MARC-145 cells with methyl-β-cyclodextrin(MβCD),a drug used to deplete cholesterol from cellular membrane,significantly reduced PRRSV infection in a dose-dependent manner.This inhibition was partially reversed by supplementing exogenous cholesterol following MβCD treatment,suggesting that the inhibition of PRRSV infection was specifically mediated by removal of cellular cholesterol.Further detailed studies showed that depletion of cellular membrane cholesterol significantly inhibited virus entry,especially virus attachment and release.These results indicate that the presence of cholesterol in the cellular membrane is a key component of PRRSV infection.

Hemagglutinin-esterase-fusion （HEF） protein of influenza C virus

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglu- tinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemaggiutinin （HA） and Neuraminidase （NA） of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post- translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF： receptor binding, esterase activity and membrane fusion.

Characterization of two monoclonal antibodies, 38F10 and 44D11, against the major envelope fusion protein of Helicoverpa armigera nucleopolyhedrovirus

The envelope fusion protein F of baculoviruses is a class I viral fusion protein which play a significant role during virus entry into insect cells. F is initially synthesized as a precursor（F_0） and then cleaved into a disulfide-linked F_1 and F_2 subunits during the process of protein maturation and secretion. To facilitate further investigation into the structure and function of F protein during virus infection, monoclonal antibodies（mAbs） against the F_2 subunit of Helicoverpa armigera nucleopolyhedrovirus（HearNPV）（Ha F） were generated. Two kinds of mAbs were obtained according to their different recognition epitopes： one kind of mAbs, as represented by 38F10,recognizes amino acid（aa） 85 to 123 of F_2 and the other kind, represented by 44D11, recognizes aa148 to 173 of F_2. Western blot and immunofluorescence assay confirmed that both of the mAbs recognized the F protein expressed in HearNPV infected cells, however, only 44D11 could neutralize HearNPV infection. The results further showed that 44D11 may not interact with a receptor binding epitope, rather it was demonstrated to inhibit syncytium formation in cells expressing the Ha F protein. The results imply that the monoclonal antibody 44D11 recognizes a region within HaF_2 that may be involved in the F-mediated membrane fusion process. 基金