Herpes Simplex Virus Entry Research Articles

Glycoprotein-Dependent and TLR2-Independent Innate Immune Recognition of Herpes Simplex Virus-1 by Dendritic Cells

Innate immune recognition is an important early event in the host response to herpes simplex virus-1 (HSV-1) infection. Dendritic cells (DC) play an important sentinel role in this recognition. Previous studies have shown that monocyte-derived DC (MDDC) respond to HSV-1 by up-regulation of costimulatory molecules and type I IFN release, but the molecular targets on the virus recognized by the DC have not been defined. In this study we show that MDDC recognize and respond to the four essential viral glycoproteins, gB, gD, and gHgL, independent of other viral proteins or nucleic acids. DC recognition of these four glycoproteins leads to the up-regulation of CD40, CD83, CD86, and HLA-DR and to the production of IFN-alpha and IL-10, but not IL-12p70. Glutaraldehyde-fixation and nonfunctional gH mutants were used to show that recognition of glycoproteins does not require membrane fusion. The nature of the recognition event was probed further by transfecting glycoproteins individually or in combination, by blocking individual proteins with Abs, or by using mutant gD constructs unable to bind to their known cognate receptors. Unexpectedly, MDDC responses were found to require expression of all four glycoproteins. Furthermore, gD mutants that cannot bind nectin-1 and/or herpesvirus entry mediator can still induce DC maturation. Finally, although HSV-1 can signal via the TLR2 receptor, this receptor does not mediate recognition of glycoproteins. Thus, the complex of the four essential HSV-1 entry glycoproteins on the cell surface can provide a target for innate immune recognition of this virus.

The Identification and Characterization of Fusogenic Domains in Herpes Virus Glycoprotein B Molecules

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV-1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post-translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated. Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.

Entry of herpesviruses into mammalian cells.

The mechanism that herpesviruses use to enter cells is one of the most complex viral entry mechanisms studied so far. This complexity seems to mount as new participants, both cellular receptors and viral glycoproteins, are identified. Recent structural work on entry glycoproteins gD and gB from herpes simplex virus (HSV) 1 has illuminated the functional roles of these glycoproteins in the process of entry. In doing so, it provided information on the mechanism of two critical steps of HSV entry: receptor-mediated activation and membrane fusion. Remarkably, it is becoming clear that herpesviruses have a lot in common with other, simpler viruses.

Amphipathic DNA Polymers are Candidate Vaginal Microbicides and Block Herpes Simplex virus Binding, Entry and Viral Gene Expression

Amphipathic DNA Polymers are Candidate Vaginal Microbicides and Block Herpes Simplex virus Binding, Entry and Viral Gene Expression

Cellular Proteasome Activity Facilitates Herpes Simplex Virus Entry at a Postpenetration Step

Herpes simplex virus (HSV) entry into cells is a multistep process that engages the host cell machinery. The proteasome is a large, ATP-dependent, multisubunit protease that plays a critical role in the maintenance of cell homeostasis. A battery of assays were used to demonstrate that proteasome inhibitors blocked an early step in HSV entry that occurred after capsid penetration into the cytosol but prior to capsid arrival at the nuclear periphery. Proteasome-dependent viral entry was not reliant on host or viral protein synthesis. MG132, a peptide aldehyde that competitively inhibits the degradative activity of the proteasome, had a reversible inhibitory effect on HSV entry. HSV can use endocytic or nonendocytic pathways to enter cells. These distinct entry routes were both dependent on proteasome-mediated proteolysis. In addition, HSV successfully entered cells in the absence of a functional host ubiquitin-activating enzyme, suggesting that viral entry is ubiquitin independent. We propose that proteasomal degradation of virion and/or host proteins is required for efficient delivery of incoming HSV capsids to the nucleus.

Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion

Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Dn, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10 min, and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.

Amphipathic DNA Polymers are Candidate Vaginal Microbicides and Block Herpes Simplex Virus Binding, Entry and Viral Gene Expression

Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity. The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored. REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma. REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Current Aspects of Innate and Adaptive Immunity in Atopic Dermatitis

Atopic dermatitis (AD) is a highly pruritic, chronic, multifactorial skin disease predisposing to bacterial and viral infections based on abnormalities of the innate and acquired immune system. The innate system quickly mobilizes an inflexible, standardized first-line response against different pathogens. Epidermal barrier dysfunction results in increased protein allergen penetration through the epidermis and predisposes to secondary skin infections. Two loss-of-function mutations in the epidermal filaggrin gene are associated with AD. Langerhans cells and inflammatory dendritic epidermal cells (IDEC) express high affinity IgE receptors, which are functional in IgE-mediated antigen presentation. Inducible antimicrobial peptides including the antiviral cathelicidin and the antibacterial beta-defensins show defective upregulation in lesional AD skin. The desmosomal protein nectin-1 is unmasked in AD lesions, thus becoming a relevant herpes simplex virus (HSV) entry receptor. Type I IFN-producing plasmacytoid dendritic cells are decreased and dysfunctional in AD skin, predisposing the patients to viral skin infections. Molluscum contagiosum virus produces a unique IL-18 binding protein to evade antiviral defense mechanisms. Innate and adaptive immunity do not simply coexist but are linked to one another in a complex network of skin immunobiology.

The multipartite system that mediates entry of herpes simplex virus into the cell

The multipartite entry-fusion system of herpes simplex virus is made of a quartet of glycoproteins-gD, gB, gH.gL-and three alternative gD receptors, herpesvirus entry mediator (HVEM), nectin1 and modified sites on heparan sulphate. This multipartite system recapitulates the basic steps of virus-cell fusion, i.e. receptor recognition, triggering of fusion and fusion execution. Specifically, in addition to serving as the receptor-binding glycoprotein, gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. In the unliganded gD the C-terminal region folds around the N-terminal region, such that gD adopts a closed autoinhibited conformation. In HVEM- and nectin1-bound gD the C-terminal region is displaced (opened conformation). gD is the tool for modification of HSV tropism, through insertion of ligands to heterologous tumour-specific receptors. It is discussed whether gD responds to the interaction with the natural and the heterologous receptors by adopting similar conformations, and whether the closed-to-open switch in conformation is a generalised mechanism of activation. A peculiar recombinant highlighted that the central Ig-folded core of gD may not encode executable functions for entry and that the 219-314 aa segment may be sufficient to trigger fusion. With respect to fusion execution, gB appears to be a prospective fusogen based on its coiled-coil trimeric structure, similar to that of another fusion glycoprotein. On the other hand, gH exhibits molecular elements typical of class 1 fusion glycoproteins, in particular heptad repeats and strong tendency to interact with lipids. Whether fusion execution is carried out by gB or gH.gL, or both glycoproteins in complex or sequentially remains to be determined.

Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane

Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

Molecular Umbrellas: a Novel Class of Candidate Topical Microbicides To Prevent Human Immunodeficiency Virus and Herpes Simplex Virus Infections

Molecular umbrella compounds may function as novel topical microbicides to prevent human immunodeficiency virus (HIV) and herpes simplex virus (HSV) infections. In a preliminary structure-activity investigation, one umbrella compound, designated Spm8CHAS, was identified which inhibited both HIV and HSV infections with no cellular toxicity. The objectives of the current studies were to define its spectrum of antiviral activity, characterize its mechanism of action, and explore the possibility of combining Spm8CHAS with HIV-specific reverse transcriptase inhibitors. Spm8CHAS inhibited infections by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical tissue explants exposed to HIV-1(BaL) in the presence of Spm8CHAS were completely protected (50% inhibitory concentration [IC(50)], 13.6 microg/ml), and transfer of virus to target T cells via migratory cells was abolished (IC(50), 3.8 microg/ml). Spm8CHAS inhibited HSV-2 infection of epithelial cells 10,000-fold if present throughout the infection. Notably, adding Spm8CHAS to cultures following HSV entry significantly reduced viral infection, indicating that the drug also acts postentry. Subsequent studies indicated that Spm8CHAS blocks cell-to-cell spread of HSV. Confocal microscopy using a fluorescently labeled analog of Spm8CHAS demonstrated that this conjugate crosses the plasma cell membrane and is transported to the nucleus. Combinations of Spm8CHAS with UC-781 or 9-[R-2-(phosphonylmethoxy)propyl] adenine monohydrate in vitro exhibited additive anti-HIV activity with preserved anti-HSV activity. The abilities of Spm8CHAS to inhibit primary isolates of HIV, block HSV infection postentry, and cross cell membranes support the development of a combination microbicide containing Spm8CHAS with an HIV-specific reverse transcriptase inhibitor to prevent both HIV and HSV infections by multiple mechanisms.

The principal neuronal gD-type 3- O-sulfotransferases and their products in central and peripheral nervous system tissues

Within the nervous system, heparan sulfate (HS) of the cell surface and extracellular matrix influences developmental, physiologic and pathologic processes. HS is a functionally diverse polysaccharide that employs motifs of sulfate groups to selectively bind and modulate various effector proteins. Specific HS activities are modulated by 3- O-sulfated glucosamine residues, which are generated by a family of seven 3- O-sulfotransferases (3-OSTs). Most isoforms we herein designate as gD-type 3-OSTs because they generate HS gD+, 3- O-sulfated motifs that bind the gD envelope protein of herpes simplex virus 1 (HSV-1) and thereby mediate viral cellular entry. Certain gD-type isoforms are anticipated to modulate neurobiologic events because a Drosophila gD-type 3-OST is essential for a conserved neurogenic signaling pathway regulated by Notch. Information about 3-OST isoforms expressed in the nervous system of mammals is incomplete. Here, we identify the 3-OST isoforms having properties compatible with their participation in neurobiologic events. We show that 3-OST-2 and 3-OST-4 are principal isoforms of brain. We find these are gD-type enzymes, as they produce products similar to a prototypical gD-type isoform, and they can modify HS to generate receptors for HSV-1 entry into cells. Therefore, 3-OST-2 and 3-OST-4 catalyze modifications similar or identical to those made by the Drosophila gD-type 3-OST that has a role in regulating Notch signaling. We also find that 3-OST-2 and 3-OST-4 are the predominant isoforms expressed in neurons of the trigeminal ganglion, and 3-OST-2/4-type 3- O-sulfated residues occur in this ganglion and in select brain regions. Thus, 3-OST-2 and 3-OST-4 are the major neural gD-type 3-OSTs, and so are prime candidates for participating in HS-dependent neurobiologic events.

Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions.

Glycoprotein B (gB), along with gD, gH, and gL, is essential for herpes simplex virus (HSV) entry. The crystal structure of the gB ectodomain revealed it to be an elongated multidomain trimer. We generated and characterized a panel of 67 monoclonal antibodies (MAbs). Eleven of the MAbs had virus-neutralizing activity. To organize gB into functional regions within these domains, we localized the epitopes recognized by the entire panel of MAbs and mapped them onto the crystal structure of gB. Most of the MAbs were directed to continuous or discontinuous epitopes, but several recognized discontinuous epitopes that showed some resistance to denaturation, and we refer to them as pseudo-continuous. Each category contained some MAbs with neutralizing activity. To map continuous epitopes, we used overlapping peptides that spanned the gB ectodomain and measured binding by enzyme-linked immunosorbent assay. To identify discontinuous and pseudocontinuous epitopes, a purified form of the ectodomain of gB, gB(730t), was cleaved by alpha-chymotrypsin into two major fragments comprising amino acids 98 to 472 (domains I and II) and amino acids 473 to 730 (major parts of domains III, IV, and V). We also constructed a series of gB truncations to augment the other mapping strategies. Finally, we used biosensor analysis to assign the MAbs to competition groups. Together, our results identified four functional regions: (i) one formed by residues within domain I and amino acids 697 to 725 of domain V; (ii) a second formed by residues 391 to 410, residues 454 to 475, and a less-defined region within domain II; (iii) a region containing residues of domain IV that lie close to domain III; and (iv) the first 12 residues of the N terminus that were not resolved in the crystal structure. Our data suggest that multiple domains are critical for gB function.

A novel role for phagocytosis-like uptake in herpes simplex virus entry

A novel role for phagocytosis-like uptake in herpes simplex virus entry

Access to Nectin Favors Herpes Simplex Virus Infection at the Apical Surface of Polarized Human Epithelial Cells

Viral entry may preferentially occur at the apical or the basolateral surfaces of polarized cells, and differences may impact pathogenesis, preventative strategies, and successful implementation of viral vectors for gene therapy. The objective of these studies was to examine the polarity of herpes simplex virus (HSV) entry using several different human epithelial cell lines. Human uterine (ECC-1), colonic (CaCo-2), and retinal pigment (ARPE-19) epithelial cells were grown on collagen-coated inserts, and the polarity was monitored by measuring the transepithelial cell resistance. Controls were CaSki cells, a human cervical cell line that does not polarize in vitro. The polarized cells, but not CaSki cells, were 16- to 50-fold more susceptible to HSV infection at the apical surface than at the basolateral surface. Disruption of the tight junctions by treatment with EGTA overcame the restriction on basolateral infection but had no impact on apical infection. No differences in binding at the two surfaces were observed. Confocal microscopy demonstrated that nectin-1, the major coreceptor for HSV entry, sorted preferentially to the apical surface, overlapping with adherens and tight junction proteins. Transfection with small interfering RNA specific for nectin-1 resulted in a significant reduction in susceptibility to HSV at the apical surface but had little impact on basolateral infection. Infection from the apical but not the basolateral surface triggered focal adhesion kinase phosphorylation and led to nuclear transport of viral capsids and viral gene expression. These studies indicate that access to nectin-1 contributes to preferential apical infection of these human epithelial cells by HSV.

The herpesvirus glycoproteins B and H·L are sequentially recruited to the receptor-bound gD to effect membrane fusion at virus entry

Four glycoproteins (gD, gB, gH, and gL) are required for herpes simplex virus entry into the cell or for cell-cell fusion in transfected cells. gD serves as the receptor-binding glycoprotein and as the trigger of fusion; the other three execute fusion between the viral envelope and the plasma and endocytic membranes or the membranes of adjacent cells and are highly conserved among members of the herpesvirus family. Details of the interaction of gD with gB, gH, and gL were not known. Here, we report that the four glycoproteins assemble into a complex initiated by the interaction of gD with its cellular receptor. gB is recruited to the gD-receptor complex next, even in the absence of gH.gL. gH.gL is recruited next, but only to the receptor-gD-gB ensemble. A complex with the composition receptor-gD-gB-gH.gL is assembled transiently with a life span of 15-30 min in cells exposed to virus but can also be found in infected cells and in cells committed to form polykaryocytes after transfection of the glycoprotein quartet. The results indicate that the complex assembly is a critical step in the process of virus entry and fusion, and that no viral protein other than those that participate in the complex itself is required for complex assembly. These findings imply critical protein-protein interactions among the quartet as herpes simplex virions enter the cells and at cell-cell fusion, define a specific order of recruitment, and place gH.gL as the last link in the process of glycoprotein recruitment to the complex.

A novel role for phagocytosis-like uptake in herpes simplex virus entry

It is becoming increasingly clear that herpesviruses can exploit the endocytic pathway to infect cells, yet several important features of this process remain poorly defined. Using herpes simplex virus-1 (HSV-1) as a model, we demonstrate that endocytosis of the virions mimic many features of phagocytosis. During entry, HSV-1 virions associated with plasma membrane protrusions followed by a phagocytosis-like uptake involving rearrangement of actin cytoskeleton and trafficking of the virions in large phagosome-like vesicles. RhoA GTPase was activated during this process and the mode of entry was cell type–specific. Clathrin-coated vesicles had no detectable role in virion trafficking as Eps15 dominant-negative mutants failed to affect HSV-1 uptake. Binding and fusion of the virion envelope with the phagosomal membrane is likely facilitated by clustering of nectin-1 (or HVEM) in phagosomes, which was observed in infected cells. Collectively, our data suggests a novel mode of uptake by which the virus can infect both professional and nonprofessional phagocytes.

Corneal Toxicity of Cell-Penetrating Peptides That InhibitHerpes simplexVirus Entry

Cell-penetrating peptides (CPPs) inhibit Herpes simplex virus entry at low micromolar concentrations and may be useful either as prophylactic or therapeutic agents for herpetic keratitis. The aim of this study was to assess the in vitro and in vivo toxicity of three CPPs-EB, TAT-C, and HOM (penetratin)-for the cornea. Incubation of primary (HK320) or immortalized (THK320) human keratocytes with the EB peptide (up to 100 microM), bHOMd (up to 200 microM), or TAT-C (up to 400 microM) resulted in no evidence of toxicity using a formazan dye-reduction assay. Similar results were obtained with a human trabecular meshwork cell line (TM-1), primary human foreskin fibroblasts (DP-9), Vero, and HeLa cells with EB and TATC. The bHOMd peptide showed some toxicity in Vero and HeLa cells, with CC50 values of 70 and 93 microM, respectively. The EB peptide did not inhibit macromolecular synthesis in Vero cells at concentrations below 150 microM, although cell proliferation was blocked at concentrations of EB above 50 microM. In vivo toxicity was assessed by applying peptides in Dulbecco's modified Eagle's medium to the cornea 4 times daily for 7 d. At concentrations 1000 times the IC50 values, the EB and bHOM peptides showed no toxicity, whereas TAT-C caused some mild eyelid swelling. Some slight epithelial cell sloughing was seen with the bKLA peptide in vivo. These results suggest that these CPPs-and EB in particular-have a favorable toxicity profile, and that further development is warranted.

Triggering and Visualizing the Aggregation and Fusion of Lipid Membranes in Microfluidic Chambers

We present a method that makes it possible to trigger, observe, and quantify membrane aggregation and fusion of giant liposomes in microfluidic chambers. Using electroformation from spin-coated films of lipids on transparent indium tin oxide electrodes, we formed two-dimensional networks of closely packed, surface-attached giant liposomes. We investigated the effects of fusogenic agents by simply flowing these molecules into the chambers and analyzing the resulting shape changes of more than 100 liposomes in parallel. We used this setup to quantify membrane fusion by several well-studied mechanisms, including fusion triggered by Ca 2+, polyethylene glycol, and biospecific tethering. Directly observing many liposomes simultaneously proved particularly useful for studying fusion events in the presence of low concentrations of fusogenic agents, when fusion was rare and probabilistic. We applied this microfluidic fusion assay to investigate a novel 30-mer peptide derived from a recently identified human receptor protein, B5, that is important for membrane fusion during the entry of herpes simplex virus into host cells. This peptide triggered fusion of liposomes at an ∼6 times higher probability than control peptides and caused irreversible interactions between adjacent membranes; it was, however, less fusogenic than Ca 2+ at comparable concentrations. Closely packed, surface-attached giant liposomes in microfluidic chambers offer a method to observe membrane aggregation and fusion in parallel without requiring the use of micromanipulators. This technique makes it possible to characterize rapidly novel fusogenic agents under well-defined conditions.