Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2).

Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

Peptide inhibitors of human HMG-CoA reductase as potential hypocholesterolemia agents.

Hypercholesterolemia may lead to obesity and cardiovascular diseases. To prevent hypercholesterolemia, many drugs have been developed while searching for better drugs to treat hypercholesterolemia has never been ended. Other than small molecule drugs, peptide drugs are gaining more visibilities in many therapeutic areas. In the present study, we employed phage-display techniques to screen peptide inhibitors against human HMG-CoA reductase. The results indicate that the tetrapeptide PMAS inhibits hHMGR effectively (IC50=68 µM), could be a lead compound to develop hypocholesterolemic agents.

A dodecapeptide (YQVTQSKVMSHR) exhibits antibacterial effect and induces cell aggregation in Escherichia coli.

Antimicrobial peptides play an important role in the innate immune response and host defense mechanism. In the present study, we employed phage display technique to screen for inhibitors which may block the phosphoenolpyruvatedependent phosphotransferase system (PTS) pathway and hence retard cell growth. The recombinant histidine-containing phosphocarrier HPr protein was prepared as the target to screen for the tight binders from the phage-displayed random peptide library Ph.D.-12. The biopanning processes were performed and the binding capabilities of the selected phage were further estimated by enzyme-linked immunosorbent assay (ELISA). The single-stranded DNAs of the 20 selected phages were isolated, sequenced, and five corresponding peptides were synthesized. Only one of the five peptides, AP1 (YQVTQSK VMSHR) was found to inhibit the growth of Escherichia coli cells efficiently (IC50~50 µM). Molecular modeling reveals that AP1 may block the EI-HPr interaction and phosphotransfer. Interestingly, AP1 was also found to induce cell aggregation in a concentration-dependent manner. Since glycogen accumulation has been attributed to biofilm formation, the effects of AP1 on the intracellular glycogen levels were measured. The results strongly indicate that the cell aggregation may be caused by the binding of peptide AP1 with HPr to block the interaction of dephosphorylated HPr with glycogen phosphorylase (GP). Because glycogen phosphorylase activity can be activated by HPr-GP interaction, the binding of AP1 to HPr would cause a decreasing rate of glycogen breakdown in M9 medium and accumulation of glycogen, which may lead to eventual cell aggregation. To the best of our knowledge, this is the first study to demonstrate that an inhibitor bound to a dephosphorylated HPr can decouple its regulatory function and induce cell aggregation.

Stability of gp41 hairpin and helix bundle assembly probed by combined stacking and circular dichroic approaches.

The envelope glycoprotein gp41 of HIV-1 undergoes structural rearrangement to form a helix hairpin during the virus-mediated fusion. Previous studies to investigate the folding and stability of hairpin did not monitor the end-to-end distance of the molecule. To directly probe the distance change, rhodamine dye was conjugated to the gp41 recombinant near the N- and C-terminal regions to detect the UV absorption and fluorescence intensity changes induced by the chemical denaturant guanidinium chloride (GdmCl). Using the singly- and doubly-labeled constructs allowed us to distinguish between the hairpin formation and protein oligomerization. A biphasic transition of helical structure for the wild type protein was revealed by circular dichroism measurements while unfolding of the hairpin occurred at 6M GdmCl. The relevance of our study to the fusion inhibitor for HIV-1 was borne out by results on the mutants at the positions within the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) regions. A monophasic transition at lower denaturant concentration was detected for the NHR mutant supporting the concept of differential stability of NHR and CHR helical structure. The conclusion that the observed unstacking of doubly-labeled variant arises principally from the intra-molecular dimers was drawn from the unstacking of the protein labeled in the loop. Remarkably, it is deduced that the hairpin is more stable than the CHR helical structure. A model for denaturation of the helix hairpin bundle was proposed from these results. The biological implications of the findings and further applications of the distance-based approach were discussed.

The fusion peptide domain is the primary membrane-inserted region and enhances membrane interaction of the ectodomain of HIV-1 gp41.

To execute the membrane fusion function, it is necessary for the fusion protein of the virus to penetrate into the hydrophobic milieu of membrane bilayer. Hence identification of the region(s) of the ectodomain of viral fusion proteins involved in the membrane insertion and their interaction with the rest of the fusion protein in the membrane would be important for the mechanistic study of membrane fusion. To this end, we examined membrane activity of the fusion peptide, and the ectodomain protein with or without the fusion peptide domain of HIV-1 gp41 by several biophysical measurements. The results revealed that the ectodomain protein containing the fusion peptide domain had higher membrane-perturbing activity and deeper membrane insertion, while the construct lacking the fusion peptide domain had much lower membrane activity. Strikingly, the N-terminal heptad repeat region was found to be induced deeper into the membrane by the fusion peptide, consistent with the role of the latter in the membrane penetration. We concluded that the fusion peptide is the only stretch of gp41 ectodomain that embeds deeply in the membrane interior in the prefusion stage. The function of fusion peptide in terms of membrane interaction and the implications of its interplay with other domains of gp41 on the membrane fusion cascade were discussed.

Identification of the LWYIK motif located in the human immunodeficiency virus type 1 transmembrane gp41 protein as a distinct determinant for viral infection.

The highly conserved LWYIK motif located immediately proximal to the membrane-spanning domain of the gp41 transmembrane protein of human immunodeficiency virus type 1 has been proposed as being important for the surface envelope (Env) glycoprotein's association with lipid rafts and gp41-mediated membrane fusion. Here we employed substitution and deletion mutagenesis to understand the role of this motif in the virus life cycle. None of the mutants examined affected the synthesis, precursor processing, CD4 binding, oligomerization, or cell surface expression of the Env, nor did they alter Env incorporation into the virus. All of the mutants, particularly the DeltaYI, DeltaIK, and DeltaLWYIK mutants, in which the indicated residues were deleted, exhibited greatly reduced one-cycle viral replication and the Env trans-complementation ability. All of these deletion mutant proteins were still localized in the lipid rafts. With the exception of the Trp-to-Ala (WA) mutant, which exhibited reduced viral infectivity albeit with normal membrane fusion, all mutants displayed loss of some or almost all of the membrane fusion ability. Although these deletion mutants partially inhibited in trans wild-type (WT) Env-mediated fusion, they were more effective in dominantly interfering with WT Env-mediated viral entry when coexpressed with the WT Env, implying a role of this motif in postfusion events as well. Both T20 and L43L peptides derived from the two gp41 extracellular C- and N-terminal alpha-helical heptad repeats, respectively, inhibited WT and DeltaLWYIK Env-mediated viral entry with comparable efficacies. Biotin-tagged T20 effectively captured both the fusion-active, prehairpin intermediates of WT and mutant gp41 upon CD4 activation. Env without the deletion of the LWYIK motif still effectively mediated lipid mixing but inhibited content mixing. Our study demonstrates that the immediate membrane-proximal LWYIK motif acts as a unique and distinct determinant located in the gp41 C-terminal ectodomain by promoting enlargement of fusion pores and postfusion activities.

Construction and characterization of insect cell-derived influenza VLP: cell binding, fusion, and EGFP incorporation.

We have constructed virus-like particles (VLPs) harboring hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1) ,and proton channel protein (M2) using baculovirus as a vector in the SF9 insect cell. The size of the expressed VLP was estimated to be ~100 nm by light scattering experiment and transmission electron microscopy. Recognition of HA on the VLP surface by the HA2-specific monoclonal antibody IIF4 at acidic pH, as probed by surface plasmon resonance, indicated the pH-induced structural rearrangement of HA. Uptake of the particle by A549 mediated by HA-sialylose receptor interaction was visualized by the fluorescent-labeled VLP. The HA-promoted cell-virus fusion activity was illustrated by fluorescence imaging on the Jurkat cells incubated with rhodamine-loaded VLP performed at fusogenic pH. Furthermore, the green fluorescence protein (GFP) was fused to NA to produce VLP with a pH-sensitive probe, expanding the use of VLP as an antigen carrier and a tool for viral tracking.

Recruitment of HIV-1 envelope occurs subsequent to lipid mixing: a fluorescence microscopic evidence.

Entry of the human immunodeficiency virus (HIV) into the target cell is initiated by fusion with the cell membrane, mediated through the envelope glycoproteins gp120 and gp41, following engagement to CD4 and the co-receptor. Previous fusion kinetics studies on the HXB2 envelope protein (Env) revealed that Env recruitment occurred at about 13 min concurrent with the lipid mixing. To resolve the temporal sequence of lipid mixing and recruitment, we employed an inhibitory assay monitored by fluorescence microscopy using a gp41 ectodomain (gp41e) fragment, which blocked Env recruitment in stark contrast to the lack of gp41e effect on the lipid mixing. In addition, to demonstrate the mode of action for the inhibition of gp41e, our results strongly suggested that lipid mixing precedes the Env recruitment because lipid mixing can proceed with Env recruitment inhibited by exogeneous gp41e molecules. Importantly, it was found that the random clustering of Env molecules on the membrane surface occurred at approximately 1 minute whereas the Env recruitment was observed at 13 minutes after the attachment of Env-expressing cell to the target cell. This > 10-fold temporal discrepancy highlights that the productive assembly of Env molecules leading to fusion requires spatio-temporal coordination of several adjacent Env trimers aggregated via directed movement.

The function of coreceptor as a basis for the kinetic dissection of HIV type 1 envelope protein-mediated cell fusion.

The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event.

Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex.

BACKGROUND: To study the organization and interaction with the fusion domain (or fusion peptide, FP) of the transmembrane domain (TMD) of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. RESULTS: The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. CONCLUSION: The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

Biophysical evidence of two docking sites of the carboxyl heptad repeat region within the amino heptad repeat region of gp41 of human immunodeficiency virus type 1.

Two HIV-1 gp41-derived peptide fusion inhibitors, T-20 and T-649, were synthesized and their binding profiles of the N-heptad repeat region (HR1) were compared to examine the molecular basis of the differential antiviral potency and viral resistance. Turbidity clearance experiments based on the overlapping 15-mer peptides derived from HR1 revealed a major binding site at the LLSGIV segment for both T-20 and T-649. Additionally, another docking site was found at the sequence encompassing the hydrophobic pocket of HR1 for T-649. Concordant results were observed from the surface plasmon resonance measurements. The binding affinity profile exhibited a major maximum around the LLSGIV motif for the two peptide fusion inhibitors while a less prominent docking region was located near the hydrophobic pocket for T-649. This bi-modal model deduced from T-20 and T-649 interaction with HR1 peptides could rationalize the failure of emergence of the fusion inhibitor-resistant virus with simultaneous mutations in each of the two binding regions, as well as the generally higher potency of T-649 against most viral strains.

pH-dependence of intermediate steps of membrane fusion induced by the influenza fusion peptide.

Membrane fusion mediated by the influenza-virus fusion protein is activated by low pH via a cascade of reactions. Some processes among them are irreversible, such as helix hairpin formation of the ectodomain, whereas others are reversible, such as exposure of the fusion peptide. Using this property, we attempted to dissect, in temporal order, different stages of the fusion reaction involving the fusion peptide by an acidic-neutral-acidic pH cycle. The fluorescence-quenching data indicated that both insertion depth and self-assembly are pH-reversible. In addition, lipid mixing assay was demonstrated to be arrested by neutral pH. By contrast, membrane leakage was shown to be irreversible with respect to pH. Our results, along with those from other studies on the pH-dependence of membrane fusion, are used to build a model for the virus-mediated fusion event from the perspective of pH-reversibility.

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements.

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK(a) values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CgammaH, whereas the deviation of pK(a) from the reference value for Glu4 and Glu8 CgammaH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.

Discovering Peptide Inhibitors of Human Squalene Synthase Through Screening the Phage-Displayed Cyclic Peptide c7c Library.

Many drugs for the treatment of hypercholesterolemia are targeting the enzymes involved in human cholesterol biosynthesis pathway. Squalene synthase, the rate-limiting enzyme located at the downstream of cholesterol synthesis pathway, has become a better candidate to develop next-generation hypocholesterolemia drugs. In the present study, we cloned and expressed the recombinant human squalene synthase (hSQS) as the lure to isolate potential peptide inhibitors from screening the conformation-constrained phage-displayed cyclic peptide c7c library. Their binding capabilities were further estimated by ELISA. Their pharmaceutical potentials were then analyzed through molecular modeling and the ADMET property evaluations. Four ennea-peptides and nine tetra-peptides were finally synthesized to evaluate their inhibitory potentials toward hSQS. The results indicate that the ennea-peptide CLSPHSMFC, tetra-peptides SMFC, CKTE, and WHQW can effectively inhibit hSQS activities (IC50 values equal to 64, 76, 87, and 90 µM, respectively). These peptides may have potentials to develop future cholesterol-lowering therapeutics. The ligand-protein interaction analysis also reveals that the inner hydrophobic pocket could be a more critical site of hSQS.

Exploration of Peptide Inhibitors of Human Squalene Synthase through Molecular Modeling and Phage Display Technique.

Many studies have demonstrated the role of elevated levels of serum cholesterol in the pathogenesis of atherosclerosis and coronary heart disease. Various drugs targeting the key enzymes involved in the cholesterol biosynthesis pathway have been investigated for the treatment of hypercholesterolemia. Human squalene synthase has been one of the most important targets for therapeutic intervention. In the present study, we used the recombinant human squalene synthase as the lure for screening the peptide inhibitors from phage-displayed random peptide library. The tightly bound phages and their derived peptides were further evaluated based on their potential binding capabilities, molecular modeling characteristics and predicted absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. Several hexa-peptides and tetra-peptides were finally synthesized to assay their inhibitory effects toward the recombinant human squalene synthase. The results demonstrated that the hexa-peptide FTACNW and tetra-peptide VACL can inhibit human squalene synthase effectively (with IC50 values near 100 µM) and may have potential to develop further as future hypocholesterolemia agents.

Effects of alterations of the amino-terminal glycine of influenza hemagglutinin fusion peptide on its structure, organization and membrane interactions.

Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface.

Kinetics study on the HIV-1 ectodomain protein quaternary structure formation reveals coupling of chain folding and self-assembly in the refolding cascade.

Entry of HIV-1 into the target cell is mediated by the envelope glycoprotein consisting of noncovalently associated surface subunit gp120 and transmembrane subunit gp41. To form a functional gp41 complex, the protein undergoes hairpin formation and self-assembly. The fusion event can be inhibited by gp41-derived peptides at nanomolar concentration and is highly dependent on the time of addition, implying a role of folding kinetics on the inhibitory action. Oligomerization of the gp41 ectodomain was demonstrated by light scattering measurements. Kinetic study by stopped-flow fluorescence and absorption measurements (i) revealed a multistate folding pathway and stable intermediates; (ii) showed a dissection of fast and slow components for early and late stages of folding, respectively, with 3 orders of magnitude difference in the time scale; (iii) showed the slow process was attributed to misfolding and unzipping of the hairpin; and (iv) showed retardation of the native hairpin formation is assumed to lead to coupling of the correctly registered hairpin and self-assembly. This coupling allows the deduction on the time scale of intrachain folding (0.1-1 s) for the protein. The folding reaction was illustrated by a free energy profile to explain the temporal dichotomy of fast and slow steps of folding as well as effective inhibition by gp41-derived peptide.

An efficient production and characterization of HIV-1 gp41 ectodomain with fusion peptide in Escherichia coli system.

We demonstrated a high level expression and purification of recombinant human immunodeficiency virus type 1 gp41 ectodomain (gp41e-FP) using glass bead approach with a final yield of 12±2mg/L bacterial culture. The proper folding of gp41e-FP encompassing the fusion peptide (FP) was ascertained by circular dichroism (CD) measurement and recognition by NC-1 antibody. The latter assay revealed stabilization of the gp41 coiled coil structure in the presence of liposome dispersion. The differential affinity of gp41e-FP and gp41e (devoid of FP) by NC-1 suggested an aggregated state for gp41e-FP and/or possible proximity of the fusion peptide domain to the coiled coil structure of gp41 ectodomain. Perfluorooctanoate (PFO)-PAGE electrophoresis experiment revealed the trimeric propensity of the recombinant gp41e-FP. In comparison to gp41e, the lipid mixing activity of gp41e-FP was two-fold higher suggesting a role of FP in promoting membrane fusion. The present approach to efficiently and quantitatively preparing the functional full-length recombinant gp41 ectodomain protein can be employed for structural and biomedical investigations and the extraction of other inclusion body-embedded recombinant proteins.

Direct solid-phase synthesis and fluorescence labeling of large, monodisperse mannosylated dendrons in a peptide synthesizer.

Fluorophore-labeled glycodendrimers have potential use in the study of carbohydrate-protein interactions by fluorescence spectroscopy and imaging methods. The current solution-phase methods for preparation of such glycoconjugates are labour intensive. On the other hand, the intrinsically more efficient solid-phase methods have been explored only at low generations. Herein we disclose a direct, expedient glycodendrimer synthesis from commercially available or easily prepared building blocks by machine-assisted solid-phase peptide synthesis (SPPS). Large, monodisperse 4th- and 5th-generation polylysine dendrons are prepared and capped with 16 and 32 mannose residues, respectively, in a single synthetic operation. Incorporation of a C-terminal lysine residue in the 4th-generation dendron allows fluorescence labelling with a number of common labels on resin, in organic solvent or in aqueous buffer, as required. A single HPLC purification is sufficient in all cases to obtain a homogeneous sample. The monodispersity of the glycodendrons is confirmed by MALDI-TOF. FITC-labeled 4th-generation glycodendron is an excellent probe for the imaging studies of mannose-receptor-mediated entry of into dendritic cells by confocal fluorescence microscopy.

The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis.

The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.