Peptide inhibitors of flavivirus entry derived from the E protein stem

Peptides derived from the "stem" of dengue virus (DV) type 2 (DV2) envelope (E) protein inhibit DV2 infectivity, targeting a late-stage fusion intermediate. We show here that stem peptides from all DV serotypes cross-inhibit DV1 to DV4 but that corresponding peptides derived from related flaviviruses do not. This failure to inhibit infection is not due to poor interaction with the E protein but rather to loss of association with the virion membrane. Residues 442 to 444 of the stem are determinants of inhibition; increasing hydrophobicity in this region increases inhibitory strength. These results support a two-step model of how stem-derived peptides inhibit viral entry.     

Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate

The mechanism of membrane fusion by “class II” viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein (E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The ∼55-aa-residue, membrane proximal “stem” can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4°C rather than 37°C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.     

Structure of a Dengue Virus Envelope Protein Late-Stage Fusion Intermediate

The final stages of dengue virus fusion are thought to occur when the membrane-proximal stem drives the transmembrane anchor of the viral envelope protein (E) toward the fusion loop, buried in the target cell membrane. Crystal structures of E have lacked this essential stem region. We expressed and crystallized soluble mutant forms of the dengue virus envelope protein (sE) that include portions of the juxtamembrane stem. Their structures represent late-stage fusion intermediates. The proximal part of the stem has both intra- and intermolecular interactions, so the chain “zips up” along the trimer seam. The penultimate interaction we detected involves the conserved residue F402, which has hydrophobic contacts with a conserved surface on domain II. These interactions do not require any larger-scale changes in trimer packing. The techniques for expression and crystallization of sE containing stem reported here may allow further characterization of the final stages of flavivirus fusion.     

The leukemia-specific fusion gene ETV6/RUNX1 perturbs distinct key biological functions primarily by gene repression

Background

ETV6/RUNX1 (E/R) (also known as TEL/AML1) is the most frequent gene fusion in childhood acute lymphoblastic leukemia (ALL) and also most likely the crucial factor for disease initiation; its role in leukemia propagation and maintenance, however, remains largely elusive. To address this issue we performed a shRNA-mediated knock-down (KD) of the E/R fusion gene and investigated the ensuing consequences on genome-wide gene expression patterns and deducible regulatory functions in two E/R-positive leukemic cell lines.

Findings

Microarray analyses identified 777 genes whose expression was substantially altered. Although approximately equal proportions were either up- (KD-UP) or down-regulated (KD-DOWN), the effects on biological processes and pathways differed considerably. The E/R KD-UP set was significantly enriched for genes included in the “cell activation”, “immune response”, “apoptosis”, “signal transduction” and “development and differentiation” categories, whereas in the E/R KD-DOWN set only the “PI3K/AKT/mTOR signaling” and “hematopoietic stem cells” categories became evident. Comparable expression signatures obtained from primary E/R-positive ALL samples underline the relevance of these pathways and molecular functions. We also validated six differentially expressed genes representing the categories “stem cell properties”, “B-cell differentiation”, “immune response”, “cell adhesion” and “DNA damage” with RT-qPCR.

Conclusion

Our analyses provide the first preliminary evidence that the continuous expression of the E/R fusion gene interferes with key regulatory functions that shape the biology of this leukemia subtype. E/R may thus indeed constitute the essential driving force for the propagation and maintenance of the leukemic process irrespective of potential consequences of associated secondary changes. Finally, these findings may also provide a valuable source of potentially attractive therapeutic targets.     

In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein

The flavivirus dengue virus (DV) infects cells through a low-pH-triggered membrane fusion reaction mediated by the viral envelope protein E. E is an elongated transmembrane protein with three domains and is organized as a homodimer on the mature virus particle. During fusion, the E protein homodimer dissociates, inserts the hydrophobic fusion loop into target membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives membrane fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with membranes to form core trimers. DI/II-membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV fusion.Dengue virus (DV) is a flavivirus that is spread by mosquitoes and causes millions of cases of disease each year worldwide (2, 9, 17). DV infection can result in dengue hemorrhagic fever, a more lethal disease that leads to ∼500,000 hospitalizations and ∼12,500 deaths per year (10, 39). DV is currently endemic in more than 100 countries, including the United States (17), and the World Health Organization estimates that about 40% of the world''s population lives in areas where dengue fever is endemic (39). As yet, there is no licensed DV vaccine or antiviral therapy. Studies of the molecular mechanisms of the virus life cycle are important to the development of new antiviral strategies.Flaviviruses such as DV are small, highly organized enveloped viruses with plus-sense single-stranded RNA genomes (reviewed in references 21 and 25). The flavivirus particle contains 3 structural proteins: a capsid protein, which associates with the genomic RNA to form the viral core, and two membrane proteins, the M protein and the membrane fusion protein E. Like many enveloped viruses, flaviviruses infect cells via endocytic uptake and a membrane fusion reaction triggered by the low pH within endosomes (38). Low-pH-triggered membrane fusion is mediated by conformational changes in the viral E protein, which converts from a prefusion E homodimer to a target membrane-inserted homotrimer. The structure of the DV E ectodomain in the prefusion form shows an elongated finger-like molecule with three domains (DI, DII, and DIII) composed primarily of β-sheets (22, 24, 42) (Fig. ​(Fig.1A;1A; see also Fig. ​Fig.7).7). The central DI is connected to DII. The distal tip of DII contains the hydrophobic fusion loop, the region of E that inserts into the target membrane during fusion. On the other side, DI connects via a short linker to DIII, an immunoglobulin-like domain. In the full-length viral E protein, DIII is followed by the stem, which contains 2 helical regions (H1 and H2) connected by a conserved sequence (CS). The stem connects to the C-terminal transmembrane (TM) anchor. The E-protein homodimer is arranged in a head-to-tail fashion, with the fusion loop on DII of each E protein hidden in a pocket formed by DI and DIII of its dimeric E partner.Open in a separate windowFIG. 1.Production and characterization of truncated DV2 E proteins. (A) Constructs used to express truncated forms of the DV2 E protein. At the top is a linear diagram of the full-length DV2 E protein, with DI indicated in red, DII in yellow, the fusion loop in green, the DI-DIII linker in cyan, DIII in dark blue, and the stem and TM regions in gray. L indicates the linker, and H1, CS, and H2 indicate the stem regions helix1, conserved sequence, and helix2, respectively. The residue numbers of the domain boundaries are listed below the diagram. The four S2 expression constructs primarily used in this work are shown in the middle rows. The E′-ST protein is truncated at residue 395 (DV2-NGC E-protein numbering), DI/II is truncated at residue 291, DI/II-L is truncated at residue 301, and the sequences are joined to the Strep or His tag (underlined) used for protein purification. The four DIII constructs are shown in the bottom rows, where LDIII comprises E residues 289 to 395, DIIIH1 residues 296 to 415, LDIIIH1 residues 289 to 415, and LDIIIH1CS residues 289 to 430. (B) Purified truncated E proteins were electrophoresed on SDS gels (left, 4 to 20% acrylamide; right, 10% acrylamide) under nonreducing conditions unless indicated and stained with Coomassie blue. The calculated mass of each protein (without modifications) is shown in kDa below each lane. DTT, dithiothreitol. (C) Sedimentation analysis of E proteins. Samples of purified E proteins were separated on sucrose sedimentation gradients in TAN buffer, pH 8.0, without detergent. Fractions were analyzed by SDS-PAGE, Western blotting, and Licor quantitation, all as described in Materials and Methods. Fraction 1 is the top of the gradient. (D) Inhibition of DV2 fusion by DIII proteins. Serial dilutions of DV2 were bound to BHK cells on ice and treated at pH 5.7 in the presence of the indicated DIII proteins at a final concentration of 50 μM or in buffer alone (control). Cells infected by virus fusion with the plasma membrane were quantitated by immunofluorescence. The data shown are the averages and standard deviations of three independent experiments.Open in a separate windowFIG. 7.Model for the steps in rearrangement of the dengue virus E protein during membrane fusion. DI, DII, and DIII are colored red, yellow, and blue, respectively. The hydrophobic fusion loop at the tip of DII is shown as a green star. The stem region is shown in gray and the TM domains in black. The virus membrane is shown in pink and the target membrane in blue. (I) At the top is shown the prefusion E-protein dimer, with the orientation looking down on the virus membrane. During the initial step of the fusion protein conformational change, the dimer dissociates upon exposure to low pH (bottom). (II to V) Side views of the trimerization reaction with the target membrane at the top. (II) The E fusion loops insert into the target membrane, and initial trimerization occurs between the DII tips. (III) Trimerization continues with contacts between DI and the β-strand exchange reaction. (IV) The DI-DIII linker inserts into the groove formed by strand exchange. DIII folds back against the core trimer, locking the linker into place. The trimer is now irreversible and stable in detergent. (V) In the final postfusion trimer, the stem has packed against the core trimer. The exact disposition of the fusion loops versus the stem and TM domains is not known, except that they are at the same end of the trimer, as shown in the model.Upon exposure to low pH, the homodimer dissociates and the E proteins insert their fusion loops into the target membrane and form very stable homotrimers (reviewed in reference 12). The structure of the DV E ectodomain trimer reveals that trimerization is mediated by dramatic domain movements (23, 26). The central region of the trimer is composed of DI and DII. DIII rotates by about 70°, folds back toward the target membrane, and packs against the grooves formed by DI and DII in the central trimer. During this refolding, part of the DI-DIII linker region inserts into a β-sheet of DI. These linker-DI rearrangements produce significant intersubunit contacts at the membrane-distal region of the trimer. The DV E protein stem region is not present in the trimer structure, but its length is sufficient to extend along the central trimer and connect with the TM domain. The final postfusion trimer thus has a hairpin-like conformation with the fusion loops and TM domains at the same end of the molecule. The pre- and postfusion structures of the alphavirus E1 protein (8, 18, 29) are very similar to those of the flavivirus E proteins, suggesting common features of membrane fusion between the two virus groups.Biogenesis of flavivirus particles occurs by budding into the endoplasmic reticulum (ER) and transit through the secretory pathway. The M protein is synthesized in the ER as a precursor protein termed prM, which forms a heterodimer with the E protein in the ER and on the nascent immature virus particle (19, 40, 41). Exposure to low pH in the trans-Golgi network mediates rearrangement of the viral envelope proteins and allows furin processing of prM to produce pr peptide and the mature M protein (32). The pr peptide remains associated with E throughout the low-pH environment of the secretory pathway, thereby protecting the virus from premature fusion until it is released from the cell (19, 40, 41). In the mature virus particle, the prefusion E homodimers are oriented tangentially to the virus membrane and form a herringbone-like pattern on the virus surface, essentially covering the virus membrane (16, 25).Thus, extensive structural information is available for both the DV E protein homodimer and the low-pH-induced E homotrimer. In contrast, the intermediates and mechanisms involved in the dramatic conformational transition from prefusion to postfusion E are relatively undefined. Recent studies of the flavivirus West Nile virus (WNV) suggest that an early fusion intermediate involves an extension of the stem region prior to dimer dissociation (15). Studies of the flavivirus tick-borne encephalitis (TBE) virus at pH 10 suggest that initial membrane insertion occurs via an E monomer (36). DV fusion and infection are inhibited by the addition of exogenous DIII during the E conformational change (20), implying that the central trimer region is formed before complete foldback of DIII. The presence of stem peptides can inhibit infection by DV and WNV, indicating the importance of stem interactions during hairpin formation (13). The dissociation of the TBE virus E dimer at low pH is dependent on a key histidine residue on DIII (H323; TBE virus numbering), which also promotes formation of the stable E trimer (5). However, studies of WNV indicate that viral E triggering is not controlled by protonation of a critical histidine residue (27). A better understanding of E-protein conformational changes during trimerization is important to define such intermediate steps and to evaluate their usefulness as targets for fusion inhibitors.Toward this end, in this study we expressed truncated forms of the DV E protein and used them to reconstitute steps in the trimerization reaction. This in vitro system allowed us to characterize the features of E protein involved in the formation of a stable central trimer and in DIII foldback. Our results suggest that monomeric DI/II proteins insert their fusion loops into target membranes and form a core trimer at either neutral or low pH. This core trimer is relatively unstable and can be stabilized by the binding of DIII, thus reconstituting hairpin formation.     

Membrane Uncoating of Intact Enveloped Viruses

Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.Enveloped virus entry at the plasma membrane includes binding of the virion to one or more receptors, changes in the virion components, membrane fusion, and membrane uncoating. The term “membrane uncoating” is being used to describe the separation of internal virion components from the viral membrane so the internal components can enter the cell. The term “uncoating” is sometimes used to mean the release of the viral genome from the capsid or other structures that have also entered the cell, but in this review, the term “membrane uncoating” will be used to represent only the separation of the virion internal contents and the viral envelope.Much of the original model of membrane fusion and uncoating was generally accepted as a result of a 1968 paper by Morgan and Howe (41). That paper provided strong evidence that Sendai virus (a paramyxovirus) entered a cell by fusion of the viral membrane with the cell plasma membrane. After membrane fusion, the virion rapidly lost its structure as the viral membrane merged with the host membrane and its components became part of the host membrane. The viral ribonucleoprotein (RNP) and internal proteins were released into the cytoplasm. This model of membrane uncoating is still generally accepted. For instance, in a 2007 virology text (24), this model was presented and illustrated with a figure from the Morgan and Howe paper. (The same figure is shown here as Fig. 2B.)Later, it was shown that Sendai viruses, which had been grown in fertilized chicken eggs, had different properties depending whether they had been harvested after growth for roughly 1 day (“early harvest”) or for several days (“late harvest”). The early-harvest viruses appear to be intact, but the late-harvest viruses have a different morphology and appear to be damaged (20, 26).This review summarizes data showing that intact early-harvest Sendai viruses uncoat quite differently from the way damaged late-harvest Sendai viruses uncoat. A model of intact paramyxovirus membrane uncoating is presented. The membrane uncoating of some other enveloped viruses that enter at the plasma membrane is compared to that described by this model.     

Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry

Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.     

Mutational analysis of the zippering reaction during flavivirus membrane fusion

The current model of flavivirus membrane fusion is based on atomic structures of truncated forms of the viral fusion protein E in its dimeric prefusion and trimeric postfusion conformations. These structures lack the two transmembrane domains (TMDs) of E as well as the so-called stem, believed to be involved in an intra- and intermolecular zippering reaction within the E trimer during the fusion process. In order to gain experimental evidence for the functional role of the stem in flavivirus membrane fusion, we performed a mutagenesis study with recombinant subviral particles (RSPs) of tick-borne encephalitis virus, which have fusion properties similar to those of whole infectious virions and are an established model for viral fusion. Mutations were introduced into the stem as well as that part of E predicted to interact with the stem during zippering, and the effect of these mutations was analyzed with respect to fusion peptide interactions with target cells, E protein trimerization, trimer stability, and membrane fusion in an in vitro liposome fusion assay. Our data provide evidence for a molecular interaction between a conserved phenylalanine at the N-terminal end of the stem and a pocket in domain II of E, which appears to be essential for the positioning of the stem in an orientation that allows zippering and the formation of a structure in which the TMDs can interact as required for efficient fusion.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.     

Structure of the reovirus membrane-penetration protein, Mu1, in a complex with is protector protein, Sigma3

Cell entry by nonenveloped animal viruses requires membrane penetration without membrane fusion. The reovirus penetration agent is the outer-capsid protein, Mu1. The structure of Mu1, complexed with its "protector" protein, Sigma3, and the fit of this Mu1(3)Sigma3(3) heterohexameric complex into the cryoEM image of an intact virion, reveal molecular events essential for viral penetration. Autolytic cleavage divides Mu1 into myristoylated Mu1N and Mu1C. A long hydrophobic pocket can receive the myristoyl group. Dissociation of Mu1N, linked to a major conformational change of the entire Mu1 trimer, must precede myristoyl-group insertion into the cellular membrane. A myristoyl switch, coupling exposure of the fatty acid chain, autolytic cleavage of Mu1N, and long-range molecular rearrangement of Mu1C, thus appears to be part of the penetration mechanism.     

Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals

Dengue virus (DV) is a mosquito-borne flavivirus that causes hemorrhagic fever in humans. In the natural infection, DV is introduced into human skin by an infected mosquito vector where it is believed to target immature dendritic cells (DCs) and Langerhans cells (LCs). We found that DV productively infects DCs but not LCs. We show here that the interactions between DV E protein, the sole mannosylated glycoprotein present on DV particles, and the C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) are essential for DV infection of DCs. Binding of mannosylated N-glycans on DV E protein to DC-SIGN triggers a rapid and efficient internalization of the viral glycoprotein. However, we observed that endocytosis-defective DC-SIGN molecules allow efficient DV replication, indicating that DC-SIGN endocytosis is dispensable for the internalization step in DV entry. Together, these results argue in favor of a mechanism by which DC-SIGN enhances DV entry and infection in cis. We propose that DC-SIGN concentrates mosquito-derived DV particles at the cell surface to allow efficient interaction with an as yet unidentified entry factor that is ultimately responsible for DV internalization and pH-dependent fusion into DCs.     

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.     

Ebolavirus delta-peptide immunoadhesins inhibit marburgvirus and ebolavirus cell entry

With the exception of Reston and Lloviu viruses, filoviruses (marburgviruses, ebolaviruses, and “cuevaviruses”) cause severe viral hemorrhagic fevers in humans. Filoviruses use a class I fusion protein, GP_1,2, to bind to an unknown, but shared, cell surface receptor to initiate virus-cell fusion. In addition to GP_1,2, ebolaviruses and cuevaviruses, but not marburgviruses, express two secreted glycoproteins, soluble GP (sGP) and small soluble GP (ssGP). All three glycoproteins have identical N termini that include the receptor-binding region (RBR) but differ in their C termini. We evaluated the effect of the secreted ebolavirus glycoproteins on marburgvirus and ebolavirus cell entry, using Fc-tagged recombinant proteins. Neither sGP-Fc nor ssGP-Fc bound to filovirus-permissive cells or inhibited GP_1,2-mediated cell entry of pseudotyped retroviruses. Surprisingly, several Fc-tagged Δ-peptides, which are small C-terminal cleavage products of sGP secreted by ebolavirus-infected cells, inhibited entry of retroviruses pseudotyped with Marburg virus GP_1,2, as well as Marburg virus and Ebola virus infection in a dose-dependent manner and at low molarity despite absence of sequence similarity to filovirus RBRs. Fc-tagged Δ-peptides from three ebolaviruses (Ebola virus, Sudan virus, and Taï Forest virus) inhibited GP_1,2-mediated entry and infection of viruses comparably to or better than the Fc-tagged RBRs, whereas the Δ-peptide-Fc of an ebolavirus nonpathogenic for humans (Reston virus) and that of an ebolavirus with lower lethality for humans (Bundibugyo virus) had little effect. These data indicate that Δ-peptides are functional components of ebolavirus proteomes. They join cathepsins and integrins as novel modulators of filovirus cell entry, might play important roles in pathogenesis, and could be exploited for the synthesis of powerful new antivirals.     

Cross-Inhibition of Chikungunya Virus Fusion and Infection by Alphavirus E1 Domain III Proteins

Alphaviruses are small enveloped RNA viruses that include important emerging human pathogens, such as chikungunya virus (CHIKV). These viruses infect cells via a low-pH-triggered membrane fusion reaction, making this step a potential target for antiviral therapies. The E1 fusion protein inserts into the target membrane, trimerizes, and refolds to a hairpin-like conformation in which the combination of E1 domain III (DIII) and the stem region (DIII-stem) pack against a core trimer composed of E1 domains I and II (DI/II). Addition of exogenous DIII proteins from Semliki Forest virus (SFV) has been shown to inhibit E1 hairpin formation and SFV fusion and infection. Here we produced and characterized DIII and DI/II proteins from CHIKV and SFV. Unlike SFV DIII, both core trimer binding and fusion inhibition by CHIKV DIII required the stem region. CHIKV DIII-stem and SFV DIII-stem showed efficient cross-inhibition of SFV, Sindbis virus, and CHIKV infections. We developed a fluorescence anisotropy-based assay for the binding of SFV DIII-stem to the core trimer and used it to demonstrate the relatively high affinity of this interaction (K_d [dissociation constant], ∼85 nM) and the importance of the stem region. Together, our results support the conserved nature of the key contacts of DIII-stem in the alphavirus E1 homotrimer and describe a sensitive and quantitative in vitro assay for this step in fusion protein refolding.     

Human Parainfluenza Virus Type 3 HN-Receptor Interaction: Effect of 4-Guanidino-Neu5Ac2en on a Neuraminidase-Deficient Variant

The envelope of human parainfluenza virus type 3 (HPF3) contains two viral glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion protein (F). HN, which is responsible for receptor attachment and for promoting F-mediated fusion, also possesses neuraminidase (receptor-destroying) activity. We reported previously that 4-guanidino-neu5Ac2en (4-GU-DANA) and related sialic acid-based inhibitors of HPF3 neuraminidase activity also inhibit HN-mediated receptor binding and fusion processes not involving neuraminidase activity. We have now examined this mechanism, as well as neuraminidase's role in the viral life cycle, using a neuraminidase-deficient HPF3 variant (C28a) and stable cell lines expressing C28a or wild-type (wt) HN. C28a, which has a wt F sequence and two point mutations in the HN gene corresponding to two amino acid changes in the HN protein, is the first HPF3 variant with insignificant neuraminidase activity. Cells expressing C28a HN did not bind erythrocytes at 4 degrees C unless pretreated with neuraminidase, but no such pretreatment was required for hemadsorption activity (HAD) at 22 or 37 degrees C. HAD was blocked by 4-GU-DANA, attesting to the ability of this compound to inhibit HN's receptor-binding activity. C28a or wt plaque enlargement, a process that involves cell-cell fusion and does not depend on virion release, is diminished by the presence of 4-GU-DANA, confirming the inhibitory effect of 4-GU-DANA on the fusogenic function of C28a HN. In C28a-infected cell monolayers, virion release and thus multicycle replication are severely restricted. This defect was corrected by supplementation of exogenous neuraminidase and also by the addition of 4-GU-DANA; neuraminidase destroys the receptors whereby newly formed C28a virions would remain attached to the cell surface, whereas 4-GU-DANA prevents the attachment itself, obviating the need for receptor cleavage. In accord with the ability of 4-GU-DANA to prevent attachment, the neuraminidase inhibitory effect of 4-GU-DANA on wt HPF3 did not diminish virion release into the medium. Thus, it is by inhibition of viral entry and syncytium formation that sialic acid analogs like 4-GU-DANA may counteract wt HPF3 infection.     

Analysis of the subunit stoichiometries in viral entry

Virions of the Human Immunodeficiency Virus (HIV) infect cells by first attaching with their surface spikes to the CD4 receptor on target cells. This leads to conformational changes in the viral spikes, enabling the virus to engage a coreceptor, commonly CCR5 or CXCR4, and consecutively to insert the fusion peptide into the cellular membrane. Finally, the viral and the cellular membranes fuse. The HIV spike is a trimer consisting of three identical heterodimers composed of the gp120 and gp41 envelope proteins. Each of the gp120 proteins in the trimer is capable of attaching to the CD4 receptor and the coreceptor, and each of the three gp41 units harbors a fusion domain. It is still under debate how many of the envelope subunits within a given trimer have to bind to the CD4 receptors and to the coreceptors, and how many gp41 protein fusion domains are required for fusion. These numbers are referred to as subunit stoichiometries. We present a mathematical framework for estimating these parameters individually by analyzing infectivity assays with pseudotyped viruses. We find that the number of spikes that are engaged in mediating cell entry and the distribution of the spike number play important roles for the estimation of the subunit stoichiometries. Our model framework also shows why it is important to subdivide the question of the number of functional subunits within one trimer into the three different subunit stoichiometries. In a second step, we extend our models to study whether the subunits within one trimer cooperate during receptor binding and fusion. As an example for how our models can be applied, we reanalyze a data set on subunit stoichiometries. We find that two envelope proteins have to engage with CD4-receptors and coreceptors and that two fusion proteins must be revealed within one trimer for viral entry. Our study is motivated by the mechanism of HIV entry but the experimental technique and the model framework can be extended to other viral systems as well.     

Site-directed antibodies against the stem region reveal low pH-induced conformational changes of the Semliki Forest virus fusion protein

The E1 envelope protein of the alphavirus Semliki Forest virus (SFV) is a class II fusion protein that mediates low pH-triggered membrane fusion during virus infection. Like other class I and class II fusion proteins, during fusion E1 inserts into the target membrane and rearranges to form a trimeric hairpin structure. The postfusion structures of the alphavirus and flavivirus fusion proteins suggest that the "stem" region connecting the fusion protein domain III to the transmembrane domain interacts along the trimer core during the low pH-induced conformational change. However, the location of the E1 stem in the SFV particle and its rearrangement and functional importance during fusion are not known. We developed site-directed polyclonal antibodies to the N- or C-terminal regions of the SFV E1 stem and used them to study the stem during fusion. The E1 stem was hidden on neutral pH virus but became accessible after low pH-triggered dissociation of the E2/E1 heterodimer. The stem packed onto the trimer core in the postfusion conformation and became inaccessible to antibody binding. Generation of the E1 homotrimer on fusion-incompetent membranes identified an intermediate conformation in which domain III had folded back but stem packing was incomplete. Our data suggest that E1 hairpin formation occurs by the sequential packing of domain III and the stem onto the trimer core and indicate a tight correlation between stem packing and membrane merger.     

Pseudorevertants of a Semliki Forest Virus Fusion-Blocking Mutation Reveal a Critical Interchain Interaction in the Core Trimer

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.Enveloped viruses contain a phospholipid bilayer that surrounds and protects the viral genome until fusion of the virus and host membranes delivers the genome into the cytoplasm. Fusion is mediated by transmembrane fusion proteins in the virus envelope. Viruses have evolved specific mechanisms to trigger membrane fusion upon interaction with the host cell (15, 42). For example, the fusion protein of the human immunodeficiency virus is triggered by receptor and coreceptor binding, while alphaviruses such as Semliki Forest virus (SFV) and flaviviruses such as dengue virus are triggered by exposure to acidic pH. The fusion trigger initiates the conversion of the fusion protein from the metastable prefusion state to the more energetically stable postfusion state (14, 15). The energy released during the refolding of the membrane fusion protein drives the merger of the viral and host membranes.Alphaviruses take advantage of the low-pH environment of the endocytic pathway to trigger membrane fusion during entry (37). E1 is the fusion protein and forms heterodimers with the E2 protein on the virus surface. These heterodimers are organized into trimers (E2/E1)₃ to form the icosahedral glycoprotein shell (21, 30, 43). Alphaviruses bind to cell surface receptors and are internalized by clathrin-mediated endocytosis and delivered to endosomes (16). Here, low pH induces E1/E2 heterodimer dissociation, E1 insertion into endosomal membranes, and the refolding of E1 to the final postfusion homotrimer conformation (16, 37). The resultant membrane fusion releases the viral RNA genome into the cytoplasm to initiate virus replication. During replication the envelope glycoproteins are translated in the endoplasmic reticulum (ER), processed through the cellular secretory pathway, and delivered to the plasma membrane, where budding of virus particles occurs (20).The alphavirus membrane fusion protein E1 and the flavivirus membrane fusion protein E are structurally related. These proteins are often referred to as class II fusion proteins to distinguish them from the class I proteins (exemplified by influenza hemagglutinin [HA] and HIV gp41) and the class III proteins (exemplified by vesicular stomatitis virus G and baculovirus gp64) (reviewed in references 15, 19, and 42). Class II fusion proteins such as the SFV E1 protein are composed almost exclusively of β-sheets organized into three domains (DI to DIII) (22, 35). There is a central DI that connects to the elongated DII containing the hydrophobic fusion loop at the tip. The other side of DI connects to DIII, followed by the stem and transmembrane domain that anchors the protein to the viral membrane. Unlike the class I and class III proteins, the alphavirus and flavivirus fusion proteins are dimers in the prefusion state and homotrimers in the postfusion state. During the prefusion to postfusion transition, DIII moves approximately 37 Å toward the target membrane-inserted fusion loop. The resulting hairpin-like conformation brings the viral and host membranes together to mediate membrane fusion (4, 13, 31) (see Fig. ​Fig.11 for the SFV E1 homotrimer structure).Open in a separate windowFIG. 1.Location of revertants in the E1 trimer. (A) The crystal structure of the postfusion E1* homotrimer (PDB entry 1RER) is shown with two chains in light gray and one chain colored as follows: DI in red, DII in yellow, DIII in blue, the fusion loop in green, the DI-DIII linker in black, and the N-terminal region of the stem in purple. The C-terminal stem connects to the transmembrane domain (neither of these is present in the crystal structure). The E1 residues discussed in this work are labeled and are represented as sticks highlighted with colors for clarity. D188 on the g-h loop in DII is shown in cyan, K176 on the DII β-strand f is in pink, and P14 on the DI β-strand C₀ is in orange. (B) A view of the central trimer interface (fusion loops pointing toward the viewer) showing the positions of D188 and K176 in the crystal structure, with colors as in panel A but with oxygen shown in red and nitrogen in blue on the stick structures. A holmium atom (not shown) is coordinated by the three inwardly pointing D188 residues. This figure was prepared using PyMol (9).Alphavirus membrane fusion is a necessary step for virus infection and occurs rapidly and efficiently with a threshold pH of ∼6.2 (reviewed in reference 16). Mutations that block trimerization prevent virus fusion and infection (18, 29). Similarly, chemical inhibition of trimerization inhibits fusion in a virus-liposome system (8). Fusion and infection are also specifically inhibited by the addition of exogenous DIII, which binds a trimeric intermediate of E1 and prevents fold-back of endogenous DIII and formation of the final postfusion trimer (25).Although formation of the E1 homotrimer is crucial to membrane fusion, little is known about the residues that regulate the overall process and steps of trimerization. The dramatic effects of local environment on the pK_a of ionizable residues make it difficult to predict the key players that initiate and drive E1 refolding, despite the fact that it takes place in a physiological window between pH values of ∼5 and 7 (37). The postfusion structures of E1 and E show that DI and DII comprise the central region of the trimer and that DIII and the stem pack against this core to form the outer layer of the trimer (4, 13, 31). It was recently shown that a truncated version of SFV E1 containing only DI and DII forms a stable core trimer with biochemical features similar to those of the full-length trimer (38). This result suggests that important interactions exist within the alphavirus core trimer.Inspection of the E1 postfusion structure identified a conserved aspartate residue, D188, located in the central trimer interface. This residue was shown to play an important role in the initial events of trimerization (29). Mutation of D188 to lysine (D188K) blocks virus fusion and infection and prevents stable trimers from forming while having no effect on E2/E1 heterodimer dissociation or E1 membrane insertion. Here, we have selected and characterized viable revertants of the D188K mutant and used them to identify an important interaction of D188 with a lysine residue on the adjoining E1 chain. This ring of salt bridges acts to stabilize the E1 core trimer and helps to drive formation of an extended trimer intermediate.(The data in this paper are from a thesis to be submitted by C. Y. Liu in partial fulfillment of the requirements for a Ph.D. in the Graduate Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, New York, NY.)