Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

AbstractParamyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process—an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1–5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described

A dual-functional paramyxovirus F protein regulatory switch segment: activation and membrane fusion

Many viral fusion–mediating glycoproteins couple α-helical bundle formation to membrane merger, but have different methods for fusion activation. To study paramyxovirus-mediated fusion, we mutated the SV5 fusion (F) protein at conserved residues L447 and I449, which are adjacent to heptad repeat (HR) B and bind to a prominent cavity in the HRA trimeric coiled coil in the fusogenic six-helix bundle (6HB) structure. These analyses on residues L447 and I449, both in intact F protein and in 6HB, suggest a metamorphic region around these residues with dual structural roles. Mutation of L447 and I449 to aliphatic residues destabilizes the 6HB structure and attenuates fusion activity. Mutation of L447 and I449 to aromatic residues also destabilizes the 6HB structure despite promoting hyperactive fusion, indicating that 6HB stability alone does not dictate fusogenicity. Thus, residues L447 and I449 adjacent to HRB in paramyxovirus F have distinct roles in fusion activation and 6HB formation, suggesting this region is involved in a conformational switch

Assembly and architecture of the EBV B cell entry triggering complex.

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion

Monomeric ephrinB2 binding induces allosteric changes in Nipah virus G that precede its full activation.

Nipah virus is an emergent paramyxovirus that causes deadly encephalitis and respiratory infections in humans. Two glycoproteins coordinate the infection of host cells, an attachment protein (G), which binds to cell surface receptors, and a fusion (F) protein, which carries out the process of virus-cell membrane fusion. The G protein binds to ephrin B2/3 receptors, inducing G conformational changes that trigger F protein refolding. Using an optical approach based on second harmonic generation, we show that monomeric and dimeric receptors activate distinct conformational changes in G. The monomeric receptor-induced changes are not detected by conformation-sensitive monoclonal antibodies or through electron microscopy analysis of G:ephrinB2 complexes. However, hydrogen/deuterium exchange experiments confirm the second harmonic generation observations and reveal allosteric changes in the G receptor binding and F-activating stalk domains, providing insights into the pathway of receptor-activated virus entry.Nipah virus causes encephalitis in humans. Here the authors use a multidisciplinary approach to study the binding of the viral attachment protein G to its host receptor ephrinB2 and show that monomeric and dimeric receptors activate distinct conformational changes in G and discuss implications for receptor-activated virus entry

H-IPSE is a pathogen-secreted host nucleus infiltrating protein (infiltrin) expressed exclusively by the Schistosoma haematobium egg stage

Urogenital schistosomiasis, caused by the parasitic trematode Schistosoma haematobium, affects over 112 million people worldwide. As with S. mansoni infections, the pathology in urogenital schistosomiasis is mainly related to the egg stage, which induces granulomatous inflammation of affected tissues. Schistosoma eggs and their secretions have been studied extensively for the related S. mansoni organism which is more amenable to laboratory studies. Indeed, we have shown that IPSE/alpha-1 (M-IPSE herein), a major protein secreted from S .mansoni eggs, can infiltrate host cells. Although M-IPSE function is unknown, its ability to translocate to their nucleus and bind DNA suggests a possible role in immune modulation of host cell tissues. Whether IPSE homologs are expressed in other Schistosome species has not been investigated. Here, we describe the cloning of two paralog genes H03-IPSE and H06-IPSE which are the ortholog of M-IPSE, from the egg-cDNA of S. haematobium. Using PCR and immunodetection, we confirmed that expression of these genes is restricted to the egg stage and female adult worms, while H-IPSE protein is only detectable in mature eggs but not adults. We show that both H03-IPSE and H06-IPSE proteins can infiltrate HTB-9 bladder cells when added exogenously to culture medium. Monopartite C-terminal NLS motifs conserved in H03-IPSE ‘SKRRRKY’ and H06-IPSE ‘SKRGRKY’ NLS motifs, are responsible for targeting the proteins to the nucleus of HTB-9 cells, as demonstrated by site directed mutagenesis and GFP tagging. Thus, S. haematobium eggs express IPSE homologs that appear to perform similar functions in infiltrating host cells

IPSE, a parasite-derived host immunomodulatory protein, is a potential therapeutic for hemorrhagic cystitis

Chemotherapy-induced hemorrhagic cystitis is characterized by bladder pain and voiding dysfunction caused by hemorrhage and inflammation. Novel therapeutic options to treat hemorrhagic cystitis are needed. We previously reported that systemic administration of the Schistosomiasis haematobium-derived protein H-IPSEH06 (IL-4-inducing principle from Schistosoma mansoni eggs), is superior to 3 doses of MESNA in alleviating hemorrhagic cystitis. Based on prior reports by others on S. mansoni IPSE (M-IPSE) and additional work by our group, we reasoned that H-IPSE mediates its effects on hemorrhagic cystitis by binding IgE on basophils and inducing IL-4 expression, promoting urothelial proliferation, and translocating to the nucleus to modulate expression of genes implicated in relieving bladder dysfunction. We speculated that local bladder injection of the S. haematobium IPSE ortholog IPSEH03, hereafter called H-IPSEH03, might be more efficacious in preventing hemorrhagic cystitis compared to systemic administration of IPSEH06. We report that H-IPSEH03, like M-IPSE and H-IPSEH06, activates IgE-bearing basophils in an NFAT reporter assay, indicating activation of the cytokine pathway. Further, H-IPSEH03 attenuates ifosfamide-induced increases in bladder wet weight in an IL-4-dependent fashion. H-IPSEH03 relieves hemorrhagic cystitis-associated allodynia and modulates voiding patterns in mice. Finally, H-IPSEH03 drives increased urothelial cell proliferation suggesting that IPSE induces bladder repair mechanisms. Taken together, H-IPSEH03 may be a potential novel therapeutic to treat hemorrhagic cystitis by basophil activation, attenuation of allodynia and promotion of urothelial cell proliferation

Phylogenomic Analyses Reveal the Evolutionary Origin of the Inhibin α-Subunit, a Unique TGFβ Superfamily Antagonist

Transforming growth factor-beta (TGFβ) homologues form a diverse superfamily that arose early in animal evolution and control cellular function through membrane-spanning, conserved serine-threonine kinases (RII and RI receptors). Activin and inhibin are related dimers within the TGFβ superfamily that share a common β-subunit. The evolution of the inhibin α-subunit created the only antagonist within the TGFβ superfamily and the only member known to act as an endocrine hormone. This hormone introduced a new level of complexity and control to vertebrate reproductive function. The novel functions of the inhibin α-subunit appear to reflect specific insertion-deletion changes within the inhibin β-subunit that occurred during evolution. Using phylogenomic analysis, we correlated specific insertions with the acquisition of distinct functions that underlie the phenotypic complexity of vertebrate reproductive processes. This phylogenomic approach presents a new way of understanding the structure-function relationships between inhibin, activin, and the larger TGFβ superfamily

Therapeutic exploitation of IPSE, a urogenital parasite-derived host modulatory protein, for chemotherapy-induced hemorrhagic cystitis

Chemotherapy-induced hemorrhagic cystitis (CHC) can be difficult to manage. Prior work suggests IL-4 alleviates ifosfamide-induced hemorrhagic cystitis (IHC), but systemically administered IL-4 causes significant side effects. We hypothesized that the Schistosoma haematobium homolog of Interleukin-4-inducing principle from Schistosoma mansoni Eggs (H-IPSE), would reduce IHC and associated bladder pathology. IPSE binds IgE on basophils and mast cells, triggering IL-4 secretion by these cells. IPSE is also an “infiltrin”, translocating into the host nucleus to modulate gene transcription. Mice were administered IL-4, H-IPSE protein or its nuclear localization sequence (NLS) mutant with or without neutralizing anti-IL-4 antibody, or MESNA, followed by ifosfamide. Bladder tissue damage and hemoglobin content were measured. Spontaneous and evoked pain, urinary frequency and gene expression were assessed. Pain behaviors were interpreted in a blinded fashion. One dose of H-IPSE was superior to MESNA and IL-4 in suppressing bladder hemorrhage in an IL-4-and NLS-dependent fashion, and comparable to MESNA in dampening ifosfamide-triggered pain behaviors in an NLS-dependent manner. H-IPSE also accelerated urothelial repair following IHC. Our work represents the first therapeutic exploitation of a uropathogen-derived host modulatory molecule in a clinically relevant bladder disease model, and indicates that IPSE may be an alternative to MESNA for mitigating CHC

IPSE, an abundant egg-secreted protein of the carcinogenic helminth Schistosoma haematobium, promotes proliferation of bladder cancer cells and angiogenesis

Background Schistosoma haematobium, the helminth causing urogenital schistosomiasis, is a known bladder carcinogen. Despite the causal link between S. haematobium and bladder cancer, the underlying mechanisms are poorly understood. S. haematobium oviposition in the bladder is associated with angiogenesis and urothelial hyperplasia. These changes may be pre-carcinogenic events in the bladder. We hypothesized that the Interleukin-4-inducing principle of Schistosoma mansoni eggs (IPSE), an S. haematobium egg-secreted “infiltrin” protein that enters host cell nuclei to alter cellular activity, is sufficient to induce angiogenesis and urothelial hyperplasia. Methods: Mouse bladders injected with S. haematobium eggs were analyzed via microscopy for angiogenesis and urothelial hyperplasia. Endothelial and urothelial cell lines were incubated with recombinant IPSE protein or an IPSE mutant protein that lacks the native nuclear localization sequence (NLS-) and proliferation measured using CFSE staining and real-time monitoring of cell growth. IPSE’s effects on urothelial cell cycle status was assayed through propidium iodide staining. Endothelial and urothelial cell uptake of fluorophore-labeled IPSE was measured. Findings: Injection of S. haematobium eggs into the bladder triggers angiogenesis, enhances leakiness of bladder blood vessels, and drives urothelial hyperplasia. Wild type IPSE, but not NLS-, increases proliferation of endothelial and urothelial cells and skews urothelial cells towards S phase. Finally, IPSE is internalized by both endothelial and urothelial cells. Interpretation: IPSE drives endothelial and urothelial proliferation, which may depend on internalization of the molecule. The urothelial effects of IPSE depend upon its NLS. Thus, IPSE is a candidate pro-carcinogenic molecule of S. haematobium. Summary Schistosoma haematobium acts as a bladder carcinogen through unclear mechanisms. The S. haematobium homolog of IPSE, a secreted schistosome egg immunomodulatory molecule, enhances angiogenesis and urothelial proliferation, hallmarks of pre-carcinogenesis, suggesting IPSE is a key pro-oncogenic molecule of S. haematobium