Viral Entry Inhibitors Targeted to the Membrane Site of Action

The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses—human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases—the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.Fusion of enveloped viruses with the host cell is a key step in viral infectivity, and interference with this process can lead to highly effective antivirals. Viral fusion is driven by specialized proteins that undergo an ordered series of conformational changes. These changes facilitate the initial, close apposition of the viral and host membranes, and they ultimately result in the formation of a fusion pore (reviewed in reference 12). The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains: the first one (HRN) adjacent to the fusion peptide and the second one (HRC) immediately preceding the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one of them, T20 (enfuvirtide), is in clinical use for HIV-1 (19). Peptides derived from the HRN and HRC regions of paramyxovirus fusion (F) proteins can interact with fusion intermediates of F (3, 20, 22, 37, 46, 49) and provide a promising antiviral strategy.The current model for class I-driven fusion postulates the existence of a so-called prehairpin intermediate, a high-energy structure that bridges the viral and cell membranes, where the HRN and the HRC are separated. The prehairpin intermediate spontaneously collapses into the postfusion structure—a six-helical bundle (6HB), with an inner trimeric coiled-coil formed by the HRN onto which the HRC folds (12, 14, 30, 40). The key to these events is the initial activation step, whereby HN triggers F to initiate the process. Structural and biophysical analyses of the paramyxovirus 6HB (30, 50, 51) suggest that inhibitors bind to the prehairpin intermediate and prevent its transition to the 6HB, thus inhibiting viral entry. The peptides bind to their complementary HR region and thereby prevent HRN and HRC from refolding into the stable 6HB structure required for fusion (3, 10, 40). The efficiency of F triggering by HN critically influences the degree of fusion mediated by F and thus the extent of viral entry (35). In addition, differences in the efficiency of triggering of the fusion process impact the efficacy of potential antiviral molecules that target intermediate states of the fusion protein (36).Paramyxoviruses cause important human illnesses, significantly contributing to global disease and mortality, ranging from lower-respiratory-tract diseases in infants caused by human parainfluenza virus types 1, 2, and 3 (HPIV1, -2, and -3) (9, 48), to highly lethal central nervous system diseases caused by the emerging paramyxoviruses HeV and NiV. No antiviral therapies or vaccines yet exist for these paramyxoviruses, and vaccines would be unlikely to protect the youngest infants. Antiviral agents, therefore, would be particularly beneficial. All paramyxoviruses possess two envelope glycoproteins directly involved in viral entry and pathogenesis: a fusion protein (F) and a receptor-binding protein (HN, H, or G). The paramyxovirus F proteins belong to the group of “class I” fusion proteins (44, 45), which also include the influenza virus hemagglutinin protein and the HIV-1 fusion protein gp120. The F protein is synthesized as a precursor protein (F0) that is proteolytically processed posttranslationally to form a trimer of disulfide-linked heterodimers (F1-F2). This cleavage event places the fusion peptide at the F1 terminus in the mature F protein and is essential for membrane fusion activity. The exact triggers that initiate a series of conformational changes in F leading to membrane fusion differ depending on the pathway the virus uses to enter the cell. In the case of HPIV, HeV, and NiV, the receptor-binding protein, hemagglutinin-neuraminidase (HN) (in HPIV3) or G (in HeV and NiV), binds to cellular surface receptors, brings the viral envelope into proximity with the plasma membrane, and activates the viral F protein. This receptor-ligand interaction is required for the F protein to mediate the fusion of the viral envelope with the host cell membrane (23, 33, 35).The HRC peptide regions of a number of paramyxoviruses, including Sendai virus, measles virus, Newcastle disease virus (NDV), respiratory syncytial virus (RSV), simian virus 5 (SV5), Hendra virus (HeV), and Nipah virus (NiV), can inhibit the infectivity of the homologous virus (17, 20, 31, 37, 47, 49, 52, 53). Recently, we showed that peptides derived from the HRC region of the F protein of HPIV3 are effective inhibitors of both HPIV and HeV/NiV fusion (31) and that, for HeV, the strength of HRC peptide binding to the corresponding HRN region correlates with the potency of fusion and infection inhibition (30). However, peptides derived from the HPIV3 F protein HRC region are more effective at inhibiting HeV/NiV fusion than HPIV3 fusion, despite a stronger homotypic HRN-HRC interaction for HPIV3 (30, 31). We showed (36) that the kinetics of fusion (kinetics of F activation) impacts sensitivity to inhibition by peptides, as is the case for HIV (39). Alterations in HPIV3 HN′s property of F activation affect the kinetics of F''s progression through its conformational changes, thus altering inhibitor efficacy. Once the extended intermediate stage of F has passed, and fusion proceeds, peptide inhibitors are ineffective. We have proposed that the design of effective inhibitors may require either targeting an earlier stage of F activation or increasing the concentration of inhibitor at the location of receptor binding, in order to enhance the access and association of the inhibitor with the intermediate-stage fusion protein (36).A substantial body of evidence supports the notion that viral fusion occurs in confined areas of the interacting viral and host membranes (26). For HIV-1, the lipid composition of the viral membrane is strikingly different from that of the host cell membrane; the former is particularly enriched in cholesterol and sphingomyelin (4, 5, 7, 8). Cholesterol and sphingolipids are often laterally segregated in membrane microdomains or “lipid rafts” (7, 11). In fact, the antiviral potency of the HIV-inhibitory HRC peptide C34 is dramatically increased by targeting it to the “lipid rafts” via the addition of a cholesterol group (16).We applied the targeting strategy based on cholesterol derivatization to paramyxoviruses, and we show here that by adding a cholesterol tag to HPIV3-derived HRC E459V (30) inhibitory peptides, we increased antiviral potency by 2 log units (50% inhibitory concentrations [IC₅₀], <2 nM). We chose to use the HPIV3-derived peptides for HeV/NiV, because we have previously shown that they are far more effective inhibitors of HeV and NiV than the homotypic peptides (30, 31). We propose that the enhanced activity resulting from the addition of a cholesterol tag is a result of the targeting of the peptide to the plasma membrane, where fusion occurs.     

Probing the Spatial Organization of Measles Virus Fusion Complexes

The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.Paramyxoviruses infect cells through fusion of the viral envelope with target cell membranes. For all members of the Paramyxovirinae subfamily, this involves the concerted action of two envelope glycoproteins, the fusion (F) and attachment (H, HN, or G, depending on the Paramyxovirinae genus) proteins. Both proteins feature short lumenal tails, a single transmembrane domain, and large ectodomains. The F protein, in type I orientation, forms homotrimers, while homodimers or homotetramers have been suggested as functional units for attachment proteins of different Paramyxovirinae subfamily members (7, 14, 28, 41, 49, 50, 66). For entry, upon receptor binding, the attachment protein is considered to initiate a series of conformational rearrangements in the metastable prefusion F protein (15, 77), which ultimately brings together transmembrane domains and fusion peptides and, thus, donor and target membranes (3, 32, 45, 53, 80).Multiple studies have demonstrated that specific interactions between compatible F and attachment proteins of paramyxovirinae are imperative for the formation of functional fusion complexes (6, 29, 36, 42, 43, 56, 75). However, the molecular nature of these interactions and the spatial organization of functional glycoprotein hetero-oligomers remain largely unknown. Individual ectodomain and partial ectodomain crystal structures have been obtained for different paramyxovirus F (13, 76, 77) and attachment (8, 14, 17, 28, 35, 79) proteins, respectively. For F, a stabilized human parainfluenza virus type 5 (HPIV5) ectodomain that is believed to represent a prefusion conformation folds into a globular head structure that is attached to the transmembrane domains through a helical stalk consisting of the membrane-proximal heptad repeat B (HR-B) domains (77). For the attachment protein, a globular head that harbors the receptor binding sites is considered to be connected to the transmembrane region through extended stalk domains (34, 78). Crystal structures of isolated head domains have been solved for several paramyxovirus attachment proteins, including measles virus (MeV) H, and reveal the six-blade propeller fold typical of sialidase structures (8, 14, 17, 28, 79). However, morbilliviruses recognize proteinaceous receptors (for MeV, the regulator of complement activation [CD46] and/or signaling lymphocytic activation molecule [SLAM], depending on the virus strain) (21, 40, 46, 51, 64, 65). X-ray data do not extend to the stalk domains, but circular dichroism analysis (78) and structure predictions (36, 78) support an α-helical coiled-coil configuration of the stalk.The nature of individual residues that engage in specific intermolecular interactions between glycoproteins of paramyxovirinae prior to refolding has been studied most extensively for the attachment protein. The stalk domains of several paramyxovirus HN proteins have been implicated in mediating specificity for their homotypic F proteins (18, 20, 43, 63, 70, 72). We have found that this extends to MeV and canine distemper virus H and, thus, to paramyxovirinae recognizing proteinaceous receptors (36), supporting the general hypothesis that F-interacting residues may reside in the stalk region of the attachment protein (30, 78).Considerably less information concerning the nature of F microdomains that mediate attachment protein specificity is available. Among the few exceptions are peptides derived from Newcastle disease virus (NDV) and Sendai virus F HR-B domains, which interact with soluble variants of the respective HN proteins in vitro (25, 67). Multiple domains have been suggested to mediate specificity of HPIV2 F for its HN (69). However, a conclusive N-glycan shielding study (43) and structural information (77, 78) argue against direct contacts between NDV F HR-B domains and HN in native glycoprotein complexes. Thus, the role of individual HPIV2 F residues in HN binding is unclear (25, 43).Building on the observation that MeV H is able to engage in productive heterotypic interactions with F proteins derived from some but not all isolates of closely related canine distemper virus, we have recently identified residues in morbillivirus F (MeV F residue 121) and H (H stalk residues 110 to 114) that interdependently contribute to physical MeV glycoprotein interaction and F triggering for fusion (36). While these residues could mediate reciprocal glycoprotein specificity through long-range effects, molecular modeling of the MeV H stalk in an α-helical conformation has posited F residue 121 at the same level above the viral envelope as H residues 110 to 114, making direct contacts structurally conceivable (36). This spatial organization of functional fusion complexes furthermore provides a comprehensive explanation for previous demonstrations of a specific role for attachment protein stalk domains of paramyxovirinae in functional and physical interactions with F (18, 43, 63, 70, 72). However, this “staggered-head” model mandates positioning the globular head of the attachment protein above the prefusion F trimer (36), as opposed to a suggested “parallel-head” alignment of the glycoproteins (31, 47). The latter is mostly based on transmission electron microscopy micrographs of viral particles apparently showing glycoprotein spikes of equal length (33). Unfortunately, these images lack the resolution for an identification of the molecular nature of the spikes (attachment or F protein) or the distinguishing between densely packaged H and F head domains of different heights and laterally aligned head domains. Indeed, a recent single-particle reconstruction based on cryo-electron microscopy images of HPIV5 particles revealed that defined spikes correspond to F in a postfusion conformation, which was interpreted as a product of possible premature F refolding (38). These two-dimensional images of heavy-metal-stained particles did not reveal F spikes in a prefusion conformation. Rather, a dense surface layer was considered to correspond to prefusion glycoprotein hetero-oligomers (38). In addition to further-advanced image reconstructions, biochemical assessment of alternative docking modes is imperative for the elucidation of the organization of functional fusion complexes of paramyxovirinae.In this study, we subjected central predictions of the hypothetical alignment models to experimental analysis. By employing carbohydrate shielding, directed mutagenesis, and variation of the length of the H stalk domain, we examined the proximity of different regions of the H stalk to F, probed a role of individual residues around the previously identified H stalk section from positions 110 to 114 in the formation of functional fusion complexes, tested the effect of varying the length of the H stalk membrane proximal and membrane distal to this section, and explored the general possibility of whether specific contacts between the prefusion F and H head domains are required for F triggering. Experimental data were interpreted in the light of a working model of MeV glycoprotein hetero-oligomers prior to receptor binding.     

Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.Sendai virus (SeV), a murine parainfluenza virus (PIV), belongs to the genus Respirovirus within the family Paramyxoviridae (33). Sendai virus is the murine counterpart of human parainfluenza virus 1 (HPIV1), and these two viruses share high sequence homology and antigenic cross-reactivity (23, 38, 58). Both Sendai virus and HPIV1 cause respiratory diseases in their hosts that range from mild to severe, with the greatest morbidity and mortality occurring in immunocompromised hosts (3, 17). In pediatric medicine, HPIV1 is an important cause of bronchiolitis, pneumonia, and laryngotracheobronchitis, or croup (11). Other members of the genus Respirovirus include human and bovine forms of PIV3 (30).Like other paramyxoviruses, Sendai virus is an enveloped, nonsegmented, negative-strand RNA virus that invades host cells by fusion (F) protein-mediated membrane fusion at the plasma membrane (33). The receptor binding protein for Sendai virus, as well as the other parainfluenza viruses, is the hemagglutinin-neuraminidase (HN) protein. During viral entry, the HN protein binds sialic acid-containing receptors on the surfaces of host cells and then triggers the F protein to refold and cause membrane fusion (34, 40). Paramyxovirus replication occurs in the cytoplasm of infected cells, where the viral nucleocapsid is formed by the encapsidation of the viral genome with the viral nucleoprotein (N), phosphoprotein (P), and the large RNA-dependent RNA-polymerase (L) protein (33). The assembly and budding of infectious parainfluenza virions from the plasma membrane are mediated largely by the matrix (M) protein, which interacts with the viral nucleocapsid and the cytoplasmic tails of the HN and F proteins (56, 63).The paramyxovirus F protein mediates both virus-cell fusion and cell-cell fusion. Similar to other class I viral fusion proteins, paramyxovirus F proteins are expressed on the surfaces of infected cells and virions as trimers that are trapped in metastable (high energy) conformations (29, 54, 71, 73). In order to become activated for membrane fusion, uncleaved F₀ precursor protein trimers must be cleaved into a fusion-capable complex formed by F₁ and F₂ subunits (55). Field isolates of Sendai virus that have a monobasic cleavage site are cleavage activated by tryptase Clara secreted from respiratory epithelial cells (32, 69) while the pantropic F1-R laboratory isolate of Sendai virus has a mutated cleavage site and is cleaved by more ubiquitously expressed proteases (41, 67). Paramyxovirus F proteins have several regions involved in F protein conformational changes during membrane fusion: a hydrophobic fusion peptide, two 4-3 heptad repeat regions (designated heptad repeat A [HRA] and HRB), a transmembrane domain, and a cytoplasmic tail. The prefusion form of the PIV5 F₀ protein has a mushroom-like shape formed by a large globular head attached to a rod-like stalk formed by the HRB region (76). Upon triggering by the HN protein, the HRB region dissociates, the HRA region springs into a coiled coil, and the fusion peptide is inserted into the target membrane (52). Membrane fusion is catalyzed by the formation of a coiled-coil hairpin structure (2, 7, 75, 78), formed by the HRA and HRB regions, that juxtaposes the membrane-interacting fusion peptide and transmembrane domains (52). We recently performed a mutational analysis on a 10-residue sequence in the HRA region of the Sendai virus F protein (37) that forms a β-strand-turn-α-helix structure in the prefusion conformation and part of a triple-stranded coiled coil in the hairpin conformation (75, 76). The mutated residues were found to play important roles in regulating the activation and membrane fusion activity of the Sendai virus F protein, showing that F protein refolding is regulated by residues that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations.Upon triggering by the HN protein, cell surface-expressed F protein trimers mediate cell-cell fusion (syncytium formation) and extend infection in a local area (55). In nonpolarized epithelial cells, virus-induced syncytium formation has long been considered a hallmark of in vitro cytopathogenesis by respiratory paramyxoviruses. However, many questions remain regarding the extent of envelope glycoprotein expression, parainfluenza virus budding, and syncytium formation at the basolateral surfaces of polarized cells (4, 77). In an in vitro model of human airway epithelium (HAE), HPIV3 has been shown to infect ciliated epithelial cells exclusively, predominantly at the apical surface, causing little virus-mediated cytopathology, no spread of the virus beyond ciliated cells, and no syncytium formation (77). As the HAE model lacks innate and adaptive immune cells, this model would not reveal the formation of syncytia involving all cell types in the respiratory tract that are present during infection, including those that play a role in the host response to infection. In immunocompetent mice, the replication of field isolates of Sendai virus is restricted to the respiratory tract, and progeny virions bud from the apical surfaces of polarized epithelial cells (68). While syncytial cell formation in the bronchiolar epithelia of mice infected with Sendai virus has been reported previously (28), the timing of giant cell formation and its contribution to the spread of the virus and the disease it induces in the respiratory tract remain unknown.To test the hypothesis that the fusogenicity of the F protein contributes to the pathogenicity of Sendai virus in mice, the natural host of this virus, we generated two recombinant Sendai viruses containing F protein mutations in the heptad repeat A region that were found previously to either increase or decrease its fusogenic activity when the F protein was expressed from plasmid DNA constructs (37). In the present study, the effects of the F protein mutations on virus replication, F protein expression, F protein cleavage, and syncytium formation were characterized in vitro. The hyperfusogenic F-L179V virus was found to induce greater morbidity and mortality in DBA/2 mice than wild-type virus, whereas the hypofusogenic and attenuated F-K180Q virus was found to be much less pathogenic. After 1 week of infection, the F-L179V virus induced more extensive interstitial inflammation and necrosis in the lungs than the wild-type virus, including a greater number of syncytial cell masses. On the other hand, the attenuated F-K180Q virus caused much less extensive inflammation than wild-type virus and did not cause observable syncytium formation in the lungs. A comparison of 50% minimal lethal dose (MLD₅₀) values, lung titers, and histopathologic changes reveals a correlation between the membrane fusion activity of the F protein and the virulence of Sendai virus in mice.     

Blue Native PAGE and Biomolecular Complementation Reveal a Tetrameric or Higher-Order Oligomer Organization of the Physiological Measles Virus Attachment Protein H

Members of the Paramyxovirinae subfamily rely on the concerted action of two envelope glycoprotein complexes, attachment protein H and the fusion (F) protein oligomer, to achieve membrane fusion for viral entry. Despite advances in X-ray information, the organization of the physiological attachment (H) oligomer in functional fusion complexes and the molecular mechanism linking H receptor binding with F triggering remain unknown. Here, we have applied an integrated approach based on biochemical and functional assays to the problem. Blue native PAGE analysis indicates that native H complexes extract predominantly in the form of loosely assembled tetramers from purified measles virus (MeV) particles and cells transiently expressing the viral envelope glycoproteins. To gain functional insight, we have established a bimolecular complementation (BiC) assay for MeV H, on the basis of the hypothesis that physical interaction of H with F complexes, F triggering, and receptor binding constitute distinct events. Having experimentally confirmed three distinct H complementation groups, implementation of H BiC (H-BiC) reveals that a high-affinity receptor-to-paramyxovirus H monomer stoichiometry below parity is sufficient for fusion initiation, that F binding and fusion initiation are separable in H oligomers, and that a higher relative amount of F binding-competent than F fusion initiation- or receptor binding-competent H monomers per oligomer is required for optimal fusion. By capitalizing on these findings, H-BiC activity profiles confirm the organization of H into tetramers or higher-order multimers in functional fusion complexes. Results are interpreted in light of a model in which receptor binding may affect the oligomeric organization of the attachment protein complex.Enveloped viruses gain access to target cells through fusion of viral and cellular membranes. This involves viral fusogenic envelope glycoprotein complexes that mediate membrane merger in a series of conformational rearrangements. For members of the Paramyxovirinae subfamily, fusion is accomplished by the concerted action of two glycoprotein complexes, the fusion (F) protein and the attachment protein (protein H, HN, or G, depending on the Paramyxovirinae genus) (21). Receptor binding by the attachment protein is thought to trigger refolding of the metastable F complex into the thermodynamically stable postfusion conformation and thus initiate the fusion event (20).Most paramyxoviruses require coexpression of homotypic envelope glycoproteins for efficient F triggering and membrane fusion (17, 45). This implicates specific protein-protein interactions between the F and the attachment protein in functional fusion complexes. All Paramyxovirinae attachment proteins form homo-oligomers. Their ectodomains are organized in a globular head domain that shows the six-blade propeller fold typical of sialidase structures (3, 5, 8, 15, 40, 49, 52) and a stalk domain connecting the head region to the transmembrane domain and short lumenal tail. Although no crystal information on the stalk domain is available, circular dichroism analyses of parainfluenza virus type 5 (PIV 5) HN (51) and structure predictions of measles virus (MeV) H and PIV 5 HN (22, 51) support an α-helical coiled-coil configuration of the stalk. Regions in the stalk have, furthermore, been implicated for several paramyxovirus HN proteins to mediate specificity for their homotypic F proteins (9, 10, 25, 45, 47). We have demonstrated that this extends to MeV H (22, 30), supporting the view that F-interacting residues may reside in the stalk region of the attachment protein (18, 30).Despite these advances, the effect of receptor binding on the attachment protein oligomer and the molecular mechanism linking receptor binding with F-protein refolding are poorly understood. MeV H head domains have been crystallized both free and complexed with soluble receptor in monomeric and dimeric forms (5, 15). Data available for attachment proteins of related Paramyxovirus family members, such as henipavirus G, and several HN proteins suggest that the tetramer (dimer of dimers) constitutes the physiological oligomer (2, 51, 52). By extension, this may equally apply to all attachment proteins of viruses of the Paramyxovirinae subfamily. Elucidating the oligomeric status of the attachment protein engaged in functional fusion complexes in situ will likely be paramount for the mechanistic understanding of paramyxovirus F triggering, given that no major conformational differences were observed between crystal structures of PIV 5 HN, human parainfluenza virus type 3 HN, henipavirus G, and MeV H solved alone or in complex with their receptor (3, 5, 8, 15, 40, 49, 52). It was, rather, hypothesized that receptor binding may affect the quaternary status of the attachment protein homo-oligomer, which could ultimately trigger F refolding (52). If a general theme applies to paramyxovirus entry, this brings into focus the question of whether a dimeric organization represents the physiological oligomer of native MeV H.Beyond the physical organization of the H oligomer, we have only begun to uncover some of the basic dynamics that govern the complex protein machineries required for membrane fusion and virus infection. For instance, it is unknown whether physical interaction of the attachment protein complex with F and induction of F triggering are separable events within an H oligomer, whether interaction of an H oligomer with multiple F complexes is required for optimal fusion activity, or even whether membrane fusion initiation requires engagement of every protein H monomer by the receptor.In the study described here, we have employed an integrated biochemical and functional approach to better understand some of the basic structural and mechanistic features of the MeV fusion complex. Blue native PAGE (BN-PAGE) analysis was used to test the organization of the native attachment protein oligomer extracted from purified viral particles and transiently transfected cells under different stringency conditions.Bimolecular protein complementation (BiC) allows the mechanistic assessment of multisubunit protein complexes. Application to the retrovirus envelope, for instance, has elucidated the interactions between HIV-1 gp120 and gp41 and the stoichiometry of the HIV-1 envelope trimer during entry (39, 50). By applying this concept to the paramyxovirus glycoprotein system, we have newly developed an H BiC (H-BiC) assay for MeV protein H that complements BN-PAGE data with functional information and probes the stoichiometric requirements for the organization of biologically active fusion complexes and the receptor-mediated initiation of fusion. Results are interpreted in light of current hypotheses regarding the possible effects of receptor binding on paramyxovirus attachment proteins.     

Mutations in the Stalk Region of the Measles Virus Hemagglutinin Inhibit Syncytium Formation but Not Virus Entry

Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.Measles virus (MV) infection requires binding of the hemagglutinin (H) protein to its cognate receptors (9, 20, 21, 29, 41) while the fusion (F) protein triggers membrane lipid mixing and fusion. The H protein is a type II transmembrane homodimeric, disulfide-linked glycoprotein (33). The F protein is a type I membrane glycoprotein that exists as a homotrimeric complex. The protein is cleaved by furin in the trans-Golgi network into a metastable heterodimer with a membrane-spanning F₁ domain and a membrane-distal F₂ domain (16). Expressed alone, neither H nor F leads to membrane fusion, and therefore, both proteins are required and have to interact for productive infection of a target cell (46). There is evidence that these interactions start within the endoplasmic reticulum (34).The H proteins of Paramyxoviridae family members have a globular head with a six-blade β-propellor structure that is responsible for receptor binding (4, 7, 13), a stalk region composed of alpha-helical coiled coils (18, 48) that anchors the complex to the plasma membrane, and a short cytoplasmic domain that can interact with the matrix (M) protein and modulate fusion (2). Given that the F protein does not interact with a receptor on the target cell but undergoes conformational changes to enable membrane fusion, it seems likely that the F protein must interact with the H protein that enables fusion (14, 19, 23, 24, 35, 47). The molecular interactions between the F and H proteins are being increasingly understood (6, 8, 24, 25, 30, 35, 42). Hummel and Bellini have described a mutation in the H glycoprotein where threonine replaced isoleucine 98, which led to loss of fusion in chronically infected cells, but the virus was not rescued (15). Corey and Iorio performed alanine-scanning mutagenesis to determine the role of specific, membrane-proximal residues in the stalk region of the H protein responsible for H-F interactions (6). Substitution of alanine for specific residues in this region altered cell-to-cell fusion and the strength of the H-F interaction in transient-transfection experiments (6). Replacement of isoleucine with alanine at position 98 reduced fusion but did not significantly alter hemadsorption, implying that binding of the mutant H protein to CD46 was not affected (6). More recently, Paal et al. showed that the H protein can tolerate significant additions to its alpha-helical coiled coils without loss of binding or fusion in transient-transfection assays (30). Although these studies confirm the importance of the interactions between the H protein stalk and the metastable F protein for enabling fusion after receptor binding, the exact steps leading to fusion are still unclear. Moreover, studies evaluating H-F interactions were performed with transient protein expression and not in the presence of the actual virus. This is potentially an important shortcoming since the M protein can modulate infection and fusion (1).     

Crystallographic Definition of the Epitope Promiscuity of the Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2F5: Vaccine Design Implications

The quest to create a human immunodeficiency virus type 1 (HIV-1) vaccine capable of eliciting broadly neutralizing antibodies against Env has been challenging. Among other problems, one difficulty in creating a potent immunogen resides in the substantial overall sequence variability of the HIV envelope protein. The membrane-proximal region (MPER) of gp41 is a particularly conserved tryptophan-rich region spanning residues 659 to 683, which is recognized by three broadly neutralizing monoclonal antibodies (bnMAbs), 2F5, Z13, and 4E10. In this study, we first describe the variability of residues in the gp41 MPER and report on the invariant nature of 15 out of 25 amino acids comprising this region. Subsequently, we evaluate the ability of the bnMAb 2F5 to recognize 31 varying sequences of the gp41 MPER at a molecular level. In 19 cases, resulting crystal structures show the various MPER peptides bound to the 2F5 Fab′. A variety of amino acid substitutions outside the ⁶⁶⁴DKW⁶⁶⁶ core epitope are tolerated. However, changes at the ⁶⁶⁴DKW⁶⁶⁶ motif itself are restricted to those residues that preserve the aspartate''s negative charge, the hydrophobic alkyl-π stacking arrangement between the β-turn lysine and tryptophan, and the positive charge of the former. We also characterize a possible molecular mechanism of 2F5 escape by sequence variability at position 667, which is often observed in HIV-1 clade C isolates. Based on our results, we propose a somewhat more flexible molecular model of epitope recognition by bnMAb 2F5, which could guide future attempts at designing small-molecule MPER-like vaccines capable of eliciting 2F5-like antibodies.Eliciting broadly neutralizing antibodies (bnAbs) against primary isolates of human immunodeficiency virus type I (HIV-1) has been identified as a major milestone to attain in the quest for a vaccine in the fight against AIDS (12, 28). These antibodies would need to interact with HIV-1 envelope glycoproteins gp41 and/or gp120 (Env), target conserved regions and functional conformations of gp41/gp120 trimeric complexes, and prevent new HIV-1 fusion events with target cells (21, 57, 70, 71). Although a humoral response generating neutralizing antibodies against HIV-1 can be detected in HIV-1-positive individuals, the titers are often very low, and virus control is seldom achieved by these neutralizing antibodies (22, 51, 52, 66, 67). The difficulty in eliciting a broad and potent neutralizing antibody response against HIV-1 is thought to reside in the high degree of genetic diversity of the virus, in the heterogeneity of Env on the surface of HIV-1, and in the masking of functional regions by conformational covering, by an extensive glycan shield, or by the ability of some conserved domains to partition to the viral membrane (24, 25, 29, 30, 38, 39, 56, 68, 69). So far, vaccine trials using as immunogens mimics of Env in different conformations have primarily elicited antibodies with only limited neutralization potency across different HIV-1 clades although recent work has demonstrated more encouraging results (4, 12, 61).The use of conserved regions on gp41 and gp120 Env as targets for vaccine design has been mostly characterized by the very few anti-HIV-1 broadly neutralizing monoclonal antibodies (bnMAbs) that recognize them: the CD4 binding-site on gp120 (bnMAb b12), a CD4-induced gp120 coreceptor binding site (bnMAbs 17b and X5), a mannose cluster on the outer face of gp120 (bnMAb 2G12), and the membrane proximal external region (MPER) of gp41 (bnMAbs 2F5, Z13 and 4E10) (13, 29, 44, 58, 73). The gp41 MPER region is a particularly conserved part of Env that spans residues 659 to 683 (HXB2 numbering) (37, 75). Substitution and deletion studies have linked this unusually tryptophan-rich region to the fusion process of HIV-1, possibly involving a series of conformational changes (5, 37, 41, 49, 54, 74). Additionally, the gp41 MPER has been implicated in gp41 oligomerization, membrane leakage ability facilitating pore formation, and binding to the galactosyl ceramide receptor on epithelial cells for initial mucosal infection mediated by transcytosis (2, 3, 40, 53, 63, 64, 72). This wide array of roles for the gp41 MPER will put considerable pressure on sequence conservation, and any change will certainly lead to a high cost in viral fitness.Monoclonal antibody 2F5 is a broadly neutralizing monoclonal anti-HIV-1 antibody isolated from a panel of sera from naturally infected asymptomatic individuals. It reacts with a core gp41 MPER epitope spanning residues 662 to 668 with the linear sequence ELDKWAS (6, 11, 42, 62, 75). 2F5 immunoglobulin G binding studies and screening of phage display libraries demonstrated that the DKW core is essential for 2F5 recognition and binding (15, 36, 50). Crystal structures of 2F5 with peptides representing its core gp41 epitope reveal a β-turn conformation involving the central DKW residues, flanked by an extended conformation and a canonical α-helical turn for residues located at the N terminus and C terminus of the core, respectively (9, 27, 45, 47). In addition to binding to its primary epitope, evidence is accumulating that 2F5 also undergoes secondary interactions: multiple reports have demonstrated affinity of 2F5 for membrane components, possibly through its partly hydrophobic flexible elongated complementarity-determining region (CDR) H3 loop, and it has also been suggested that 2F5 might interact in a secondary manner with other regions of gp41 (1, 10, 23, 32, 33, 55). Altogether, even though the characteristics of 2F5 interaction with its linear MPER consensus epitope have been described extensively, a number of questions persist about the exact mechanism of 2F5 neutralization at a molecular level.One such ambiguous area of the neutralization mechanism of 2F5 is investigated in this study. Indeed, compared to bnMAb 4E10, 2F5 is the more potent neutralizing antibody although its breadth across different HIV-1 isolates is more limited (6, 35). In an attempt to shed light on the exact molecular requirements for 2F5 recognition of its primary gp41 MPER epitope, we performed structural studies of 2F5 Fab′ with a variety of peptides. The remarkable breadth of possible 2F5 interactions reveals a somewhat surprising promiscuity of the 2F5 binding site. Furthermore, we link our structural observations with the natural variation observed within the gp41 MPER and discuss possible routes of 2F5 escape from a molecular standpoint. Finally, our discovery of 2F5''s ability to tolerate a rather broad spectrum of amino acids in its binding, a spectrum that even includes nonnatural amino acids, opens the door to new ways to design small-molecule immunogens potentially capable of eliciting 2F5-like neutralizing antibodies.     

Identification of Key Residues in Virulent Canine Distemper Virus Hemagglutinin That Control CD150/SLAM-Binding Activity

Morbillivirus cell entry is controlled by hemagglutinin (H), an envelope-anchored viral glycoprotein determining interaction with multiple host cell surface receptors. Subsequent to virus-receptor attachment, H is thought to transduce a signal triggering the viral fusion glycoprotein, which in turn drives virus-cell fusion activity. Cell entry through the universal morbillivirus receptor CD150/SLAM was reported to depend on two nearby microdomains located within the hemagglutinin. Here, we provide evidence that three key residues in the virulent canine distemper virus A75/17 H protein (Y525, D526, and R529), clustering at the rim of a large recessed groove created by β-propeller blades 4 and 5, control SLAM-binding activity without drastically modulating protein surface expression or SLAM-independent F triggering.Paramyxoviruses are enveloped nonsegmented negative-strand RNA viruses that inject their genetic information into target cells by fusing their lipid envelope with the plasma membrane of the host cell at a neutral pH. Plasma membrane fusion activity is achieved by the concerted action of two viral membrane-bound glycoproteins. The attachment protein (hemagglutinin [H], hemagglutinin-neuraminidase [HN], or attachment [G], depending on the viral genus) is thought to bind a host cell surface receptor, in turn activating the fusion (F) protein, which will then undergo large-scale structural rearrangements, leading to plasma membrane fusion activity (9, 10, 19). In addition, both viral surface glycoproteins may mediate fusion activity between two contacting neighboring cells (22, 27). Virus-induced cell-cell fusion activity eventually leads to multinucleated cell formation (also termed syncytium formation) and, ultimately, to cell lysis.The crystal structure of the measles virus hemagglutinin (MeV-H) has recently become available (3, 7, 8). Interestingly, the overall β-propeller structure consisting of six β-sheets was well conserved compared to already determined paramyxovirus HN structures (4, 12, 29). The canine distemper virus H (CDV-H) protein has a short N-terminal cytoplasmic tail followed by a transmembrane domain and a large C-terminal ectodomain (1). It is suggested that the ectodomain consists of a stalk region with an α-helical coiled-coil configuration (13, 28) that supports a globular head domain containing the receptor recognition site and antigenic regions of the protein (11).Recently, site-directed mutagenesis aimed at identifying residues throughout the MeV-H ectodomain that might selectively control membrane fusion activity in a receptor-dependent manner (CD150/SLAM, CD46, or a yet-unidentified putative epithelial cell receptor [EpR]) was conducted. Indeed, four key residues, located in two connected microdomains (site 1 and site 2) on MeV-H globular head β-propeller blade 5, were necessary to uphold SLAM-dependent fusogenicity. Mutations in each one of the four amino acids resulted in a selective inhibition of SLAM-dependent fusion activity (H-SLAM-blind; HSB [25]). Interestingly, the latter quartet of residues were subsequently demonstrated not to be involved in SLAM-binding activity but presumably were involved in controlling SLAM-dependent F triggering (14). An additional residue (isoleucine 194), located within MeV-H β-propeller blade 6 but in contact with site 2, was next shown to govern interaction with the universal morbillivirus SLAM receptor (14). Consequently, the corresponding residues of both microdomains were mutated in the H protein of the virulent CDV strain 5804P and were also demonstrated to control SLAM-dependent fusion activity (24), although for CDV, full ablation of fusion activity required the substitutions in both microdomains and in two additional neighboring amino acids (CDV-H residues in site 1, D526, I527, S528, and R529; in site 2, Y547 and T548). Moreover, using a CDV-H 3D homology model, the two microdomains were demonstrated to be in very close proximity to one another (compared to those of MeV-H) but not in direct contact (24). Subsequently, a recombinant CDV bearing a SLAM-blind H protein was reported to be completely attenuated in ferrets, a phenotype associated with reduced immunosuppression and lack of neurovirulence (26). However, the precise molecular mechanisms sustaining HSB-dependent lack of fusion support activity was not elucidated and remains to be determined.     

Activation of Natural Killer Cells by Newcastle Disease Virus Hemagglutinin-Neuraminidase

The avian paramyxovirus Newcastle disease virus (NDV) selectively replicates in tumor cells and is known to stimulate T-cell-, macrophage-, and NK cell-mediated responses. The mechanisms of NK cell activation by NDV are poorly understood so far. We studied the expression of ligand structures for activating NK cell receptors on NDV-infected tumor cells. Upon infection with the nonlytic NDV strain Ulster and the lytic strain MTH-68/H, human carcinoma and melanoma cells showed enhanced expression of ligands for the natural cytotoxicity receptors NKp44 and NKp46, but not NKp30. Ligands for the activating receptor NKG2D were partially downregulated. Soluble NKp44-Fc and NKp46-Fc, but not NKp30-Fc, chimeric proteins bound specifically to NDV-infected tumor cells and to NDV particle-coated plates. Hemagglutinin-neuraminidase (HN) of the virus serves as a ligand structure for NKp44 and NKp46, as indicated by the blockade of binding to NDV-infected cells and viral particles in the presence of anti-HN antibodies and by binding to cells transfected with HN cDNA. Consistent with the recognition of sialic acid moieties by the viral lectin HN, the binding of NKp44-Fc and NKp46-Fc was lost after desialylation. NKp44- and NKp46-CD3ζ lacZ-inducible reporter cells were activated by NDV-infected cells. NDV-infected tumor cells stimulated NK cells to produce increased amounts of the effector lymphokines gamma interferon and tumor necrosis factor alpha. Primary NK cells and the NK line NK-92 lysed NDV-infected tumor cells with enhanced efficiency, an effect that was eliminated by the treatment of target cells with the neuraminidase inhibitor Neu5Ac2en. These results suggest that direct activation of NK cells contributes to the antitumor effects of NDV.Virulent strains of Newcastle disease virus (NDV) infect domestic poultry and other birds, causing a rapidly spreading viral disease that affects the alimentary and respiratory tracts as well as the central nervous system (55). In humans, however, NDV is well tolerated (17, 18). Other than mild fever for a day, only a few adverse effects have been reported. NDV, also known as avian paramyxovirus 1, is an enveloped virus containing a negative-sense, single-stranded RNA genome which codes for six proteins in the order (from 3′ to 5′) of nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase (HN), and large polymerase protein (19). There are many different strains of NDV, classified as either lytic or nonlytic for different types of cells. Lytic and nonlytic NDV strains both replicate much more efficiently in human cancer cells than they do in most normal human cells (43). Viruses of both strain types have been investigated as potential anticancer agents (30, 49, 52). The NDV strains that have been evaluated most widely for the treatment of cancer are 73-T, MTH-68, and Ulster (1, 7, 11, 17, 18, 53, 54, 56, 71).Initial binding of NDV to a host cell takes place through the interaction of HN molecules in the virus coat with sialic acid-containing molecules on the cell surface (31). NDV neuraminidase has strict specificity for the hydrolysis of the NeuAc-α2,3-Gal linkage, with no hydrolysis of the NeuAc-α2,6-Gal linkage (41).NDV infection of tumor cells not only improves T-cell responses (53, 58, 68), but has also been reported to vigorously stimulate innate immune responses. In the course of NDV infection, large amounts of alpha interferon (IFN-α) are released (68) and in turn activate dendritic cells and NK cells and polarize, in concert with interleukin-12 (IL-12), toward a Th1 T-cell response (33, 44, 47). In addition, NDV induces antitumor cytotoxicity in murine macrophages which produce increased amounts of tumor necrosis factor alpha (TNF-α) and nitric oxide (51, 60) and in human monocytes through the induction of TRAIL (64). Little is known about the NDV-mediated activation of NK cells. The coincubation of peripheral blood mononuclear cells with NDV was shown previously to stimulate NK-mediated cytotoxicity (70). Enhanced cytotoxicity correlates with the induction of IFN-α (70). It is not known, however, whether NDV-infected cells can directly activate NK cells and, if so, which molecular interactions are involved.The cytolytic activity of NK cells against virus-infected or tumor cells is regulated by the engagement of activating or inhibitory NK cell surface receptors, the actions of cytokines, and cross talk with other immune cells (32, 39). Most inhibitory receptors recognize particular major histocompatibility complex (MHC) class I alleles and thereby ensure the tolerance of NK cells against self antigens (38). Activating receptors on human NK cells include CD16; NKG2D; the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46; as well as NKp80; DNAM-1; and various stimulatory coreceptors (32).NCR are important activating receptors for the antitumor and antiviral activities of NK cells (5, 32, 37). Heparan sulfate has been discussed previously as a cellular ligand for NKp46, NKp44, and NKp30 (9, 26, 27), and nuclear factor BAT3, which can be released from tumor cells under stress conditions, has been described as a cellular ligand for NKp30 (42). Ligands for NKp30 and NKp44 can be detected on the surfaces and in the intracellular compartments of several kinds of tumor cells (10). Moreover, a number of pathogen-derived NCR ligands have been reported. The hemagglutinin protein of influenza virus and the HN of Sendai virus can bind to NKp46 and NKp44 and activate NK cells (3, 24, 34). The pp65 protein of human cytomegalovirus has been shown to bind NKp30 and inhibit its function (4). Human immunodeficiency virus, vaccinia virus, and herpes simplex virus have also been shown to upregulate the expression of cellular NCR ligands in infected cells (13, 14, 62). The Plasmodium falciparum erythrocyte membrane protein 1 is involved in the NCR-mediated NK cell attack against infected erythrocytes (36). Furthermore, NKp46 recognizes cells infected with mycobacteria (22, 61), and NKp44 was recently reported to directly bind to the surfaces of mycobacteria and other bacteria (21).In this study, we investigated the expression of ligand structures for NCR and NKG2D on NDV-infected cells. We demonstrate that NDV HN proteins which are strongly expressed on NDV-infected tumor cells function as activating ligand structures for NKp44 and NKp46 but that cellular ligands for NKG2D are partially downregulated during NDV infection.     

Respiratory Syncytial Virus Grown in Vero Cells Contains a Truncated Attachment Protein That Alters Its Infectivity and Dependence on Glycosaminoglycans

Human respiratory syncytial virus (RSV) contains a heavily glycosylated 90-kDa attachment glycoprotein (G). Infection of HEp-2 and Vero cells in culture depends largely on virion G protein binding to cell surface glycosaminoglycans (GAGs). This GAG-dependent phenotype has been described for RSV grown in HEp-2 cells, but we have found that it is greatly reduced by a single passage in Vero cells. Virions produced from Vero cells primarily display a 55-kDa G glycoprotein. This smaller G protein represents a post-Golgi compartment form that is lacking its C terminus, indicating that the C terminus is required for GAG dependency. Vero cell-grown virus infected primary well-differentiated human airway epithelial (HAE) cell cultures 600-fold less efficiently than did HEp-2 cell-grown virus, indicating that the C terminus of the G protein is also required for virus attachment to this model of the in vivo target cells. This reduced infectivity for HAE cell cultures is not likely to be due to the loss of GAG attachment since heparan sulfate, the primary GAG used by RSV for attachment to HEp-2 cells, is not detectable at the apical surface of HAE cell cultures where RSV enters. Growing RSV stocks in Vero cells could dramatically reduce the initial infection of the respiratory tract in animal models or in volunteers receiving attenuated virus vaccines, thereby reducing the efficiency of infection or the efficacy of the vaccine.Human respiratory syncytial virus (RSV) is a negative-sense, single-stranded RNA virus in the family Paramyxoviridae, subfamily Pneumovirinae. RSV causes mild respiratory disease in all age groups, but the disease can be severe or fatal in infants and the elderly (4, 9, 11). Initial attempts to produce a killed vaccine were not successful, resulting instead in enhanced disease upon infection (26, 41). Efforts to produce a live attenuated vaccine are ongoing (6, 7, 51).RSV produces three glycoproteins which are important for infection. The largest glycoprotein (G) is involved in attachment to the host cell (35), the fusion (F) glycoprotein mediates virion membrane fusion with the target cell membrane (2), and the small hydrophobic (SH) glycoprotein may attenuate apoptosis (15). The F protein is the only glycoprotein that is absolutely required for infection of cultured immortalized cells (27, 45) and syncytium formation, the most obvious cytopathic effect of RSV in immortalized cell culture. Although the G protein is not absolutely required for infection, it enhances infection and syncytium formation (45). The G protein attaches to cultured, immortalized cell lines (35) primarily via glycosaminoglycans (GAGs) on the cell surface (13, 22, 23, 30). GAGs are repeating disaccharide units of hexuronic acid and hexosamine that form unbranched polysaccharide chains and are found on the surface of most mammalian cells. The GAG type that appears most important for RSV infection of HEp-2 cells is heparan sulfate (HS) (23, 30).The G protein is a type II integral membrane protein with its N terminus on the cytoplasmic side of the membrane and its C terminus as the extracellular ectodomain (49). An unglycosylated region in the center of the protein contains four cysteines held together by disulfide bonds in a cysteine noose (19, 24, 33), followed, to the C-terminal side, by a predicted heparin-binding domain (HBD) (12, 13). The 32-kDa G protein, while in the endoplasmic reticulum (ER), is modified by the addition of multiple N-linked carbohydrate chains, depending on the strain. These N-linked additions would increase the molecular mass of G to 45 to 60 kDa. Previous reports have found G protein forms of this size in cells and in virions at low levels (5, 20, 21, 50). All of these reports suggest that these smaller forms of the G protein are partially glycosylated processing intermediates.Maturation of the N-linked carbohydrates of the G protein occurs in the Golgi compartment, where a large number of O-linked carbohydrate chains are added, resulting in an 84- to 92-kDa mature protein (14, 32, 35, 49). This size variation of the G protein is probably due, in part, to the difficulty in sizing heavily glycosylated molecules and variations in molecular mass markers.The G protein shares no homology with the glycoproteins of paramyxoviruses outside the Pneumovirinae subfamily. The high serine and threonine content and the high O-linked glycosylation levels are similar to those found in mucins. The amount of O-linked glycosylation is partially dependent on the cell type used to produce the virus (18).In the present study, we examined virus produced in HEp-2 and Vero cells, which are both commonly used to grow RSV in the laboratory, for dependence on GAGs by the ability to infect cells expressing GAG or deficient in GAG expression. We also examined the ability of the viruses to infect primary, well-differentiated human airway epithelial (HAE) cell cultures. In both systems, infectivity was greatly dependent upon the cell line used to grow the virus. Biochemical characterization of purified virus grown in these two cell lines revealed a smaller form of the RSV G protein in virions from Vero cells. Using C terminus-specific antibodies and a six-His tag at the C terminus of the G protein, we determined that the smaller G protein form was lacking its C terminus. These results highlight the importance of the C-terminal portion of the G protein and suggest that the cell line used to produce a virus can alter its infectivity.     

Neutralization of Human Respiratory Syncytial Virus Infectivity by Antibodies and Low-Molecular-Weight Compounds Targeted against the Fusion Glycoprotein

Human respiratory syncytial virus (HRSV) fusion (F) protein is an essential component of the virus envelope that mediates fusion of the viral and cell membranes, and, therefore, it is an attractive target for drug and vaccine development. Our aim was to analyze the neutralizing mechanism of anti-F antibodies in comparison with other low-molecular-weight compounds targeted against the F molecule. It was found that neutralization by anti-F antibodies is related to epitope specificity. Thus, neutralizing and nonneutralizing antibodies could bind equally well to virions and remained bound after ultracentrifugation of the virus, but only the former inhibited virus infectivity. Neutralization by antibodies correlated with inhibition of cell-cell fusion in a syncytium formation assay, but not with inhibition of virus binding to cells. In contrast, a peptide (residues 478 to 516 of F protein [F478-516]) derived from the F protein heptad repeat B (HRB) or the organic compound BMS-433771 did not interfere with virus infectivity if incubated with virus before ultracentrifugation or during adsorption of virus to cells at 4°C. These inhibitors must be present during virus entry to effect HRSV neutralization. These results are best interpreted by asserting that neutralizing antibodies bind to the F protein in virions interfering with its activation for fusion. Binding of nonneutralizing antibodies is not enough to block this step. In contrast, the peptide F478-516 or BMS-433771 must bind to F protein intermediates generated during virus-cell membrane fusion, blocking further development of this process.Human respiratory syncytial virus (HRSV), a member of the Pneumovirus genus of the Paramyxoviridae family, is the main cause of severe lower respiratory tract infections in very young children (36), and it is a pathogen of considerable importance in the elderly (24, 26) and in immunocompromised adults (22). Currently, there is no effective vaccine against the virus although it is known that passive administration of neutralizing antibodies to individuals at high risk is an effective immunoprophylaxis (37, 38).The HRSV genome is a single-stranded negative-sense RNA molecule of approximately 15 kb that encodes 11 proteins (16, 53). Two of these proteins are the main surface glycoproteins of the virion. These are (i) the attachment (G) protein, which mediates virus binding to cells (44), and (ii) the fusion (F) protein, which promotes both fusion of the viral and cell membranes at the initial stages of the infectious cycle and fusion of the membrane of infected cells with those of adjacent cells to form characteristic syncytia (72). These two glycoproteins are the only targets of neutralizing antibodies either induced in animal models (19, 63, 65, 70) or present in human sera (62).The G protein is a highly variable type II glycoprotein that shares neither sequence identity nor structural features with the attachment protein of other paramyxoviruses (75). It is synthesized as a precursor of about 300 amino acids (depending on the strain) that is modified posttranslationally by the addition of a large number of N- and O-linked oligosaccharides and is also palmitoylated (17). The G protein is oligomeric (probably a homotetramer) (23) and promotes binding of HRSV to cell surface proteoglycans (35, 40, 49, 67). Whether this is the only interaction of G with cell surface components is presently unknown.The F protein is a type I glycoprotein that is synthesized as an inactive precursor of 574 amino acids (F0) which is cleaved by furin during transport to the cell surface to yield two disulfide-linked polypeptides, F2 from the N terminus and F1 from the C terminus (18). Like other viral type I fusion proteins, the mature F protein is a homotrimer which is in a prefusion, metastable, conformation in the virus particle. After fusion, the F protein adopts a highly stable postfusion conformation. Stability of the postfusion conformation is determined to great extent by two heptad repeat (HR) sequences, HRA and HRB, present in the F1 chain. Mixtures of HRA and HRB peptides form spontaneously heterotrimeric complexes (43, 51) that assemble in six-helix bundles (6HB), consisting of an internal core of three HRA helices surrounded by three antiparallel HRB helices, as determined by X-ray crystallography (79).The three-dimensional (3D) structure of the HRSV F protein has not been solved yet. Nevertheless, the structures of the pre- and postfusion forms of two paramyxovirus F proteins have revealed substantial conformational differences between the pre- and postfusion conformations (77, 78). The present hypothesis about the mechanism of membrane fusion mediated by paramyxovirus F proteins proposes that, following binding of the virus to the cell surface, the prefusion form of the F glycoprotein is activated, and membrane fusion is triggered. The F protein experiences then a series of conformational changes which include the exposure of a hydrophobic region, called the fusion peptide, and its insertion into the target membrane. Subsequent refolding of this intermediate leads to formation of the HRA and HRB six-helix bundle, concomitant with approximation of the viral and cell membranes that finally fuse, placing the fusion peptide and the transmembrane domain in the same membrane (4, 20). The formation of the 6HB and the associated free energy change are tightly linked to the merger of the viral and cellular membranes (60).Antibodies play a major role in protection against HRSV. Animal studies have demonstrated that immunization with either F or G glycoproteins induces neutralizing antibodies and protects against a viral challenge (19, 63, 70). Furthermore, transfer of these antibodies (31, 56) or of anti-F or anti-G monoclonal antibodies (MAbs) protects mice, cotton rats, or calves against either a human or bovine RSV challenge, respectively (65, 68, 73). Likewise, infants at high risk of severe HRSV disease are protected by the prophylactic administration of immunoglobulins with high anti-HRSV neutralizing titers (33). Finally, a positive correlation was found between high titers of serum neutralizing antibodies and protection in adult volunteers challenged with HRSV (34, 74), while an inverse correlation was found between high titers of neutralizing antibodies and risk of infection in children (29) and in the elderly (25).Whereas all the anti-G monoclonal antibodies reported to date are poorly neutralizing (1, 28, 48, 71), some anti-F monoclonal antibodies have strong neutralization activity (1, 3, 5, 28, 46). It is believed that HRSV neutralization by anti-G antibodies requires simultaneous binding of several antibodies to different epitopes, leading to steric hindrance for interaction of the G glycoprotein with the cell surface. Indeed, it has been shown that neutralization is enhanced by mixtures of anti-G monoclonal antibodies (1, 50), mimicking the effect of polyclonal anti-G antibodies. In contrast, highly neutralizing anti-F monoclonal antibodies do not require cooperation by other antibodies to block HRSV infectivity efficiently (1).In addition to neutralizing antibodies, other low-molecular-weight compounds directed against the F protein are potent inhibitors of HRSV infectivity. Synthetic peptides that reproduce sequences of heptad repeat B inhibit both membrane fusion promoted by the F protein and HRSV infectivity (42). Also, other small molecules obtained by chemical synthesis have been shown to interact with F protein and inhibit HRSV infectivity. These HRSV entry inhibitors have been the topic of intense research in recent years (55).This study explores the mechanisms of HRSV neutralization by different inhibitors of membrane fusion, including anti-F monoclonal antibodies, an HRB peptide, and the synthetic compound BMS-433771 (13-15). The results obtained indicate that antibodies and low-molecular-weight compounds block membrane fusion at different stages during virus entry.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.