FIG 1 - uploaded by Birgit Bradel-Tretheway
Content may be subject to copyright.
FLAG tag insertion in the F 2 ectodomain and fusion phenotypes. (A) Schematic representation of the proteolytically cleaved NiV and HeV F (NF and HF). F 1 and F 2 subunits are held together by a disulfide bond. A FLAG tag was inserted just N terminal to the cathepsin L cleavage site (Cat L) located at the C terminus of F 2 , an extracellular portion of the F head domain. An AU1 tag, inserted at the intracellular C terminus of F (type I transmembrane protein), has been previously used for the detection of NF and HF (8, 11, 12, 19). FP depicts the location of the fusion peptide inserted into the target cell membrane during membrane fusion. The comparative sequences surrounding the cleavage site are shown for NiV, HeV, and CDV F glycoproteins. The N and C termini (NH 2 -and -COOH, respectively), transmembrane (TM), and cytoplasmic tail (CT) domains are indicated. (B) HEK293T cells were transfected with NF or HF expression plasmids in combination with either NG or HG. F expression constructs harbored an extracellular FLAG tag, an intracellular AU1 tag, or both (FLAG plus AU1). Sixteen to 20 hpt, cells were fixed with 1% paraformaldehyde and nuclei inside syncytia were counted (4 or more nuclei per multinucleated cell were considered a syncytium). Syncytial nuclei were counted and added within each microscopic field (200), and five microscopic fields were counted for each transfection. The results were normalized to NF (FLAG)-NG fusion, which was set at 100% (dotted line). A one-way ANOVA statistical analysis was performed, followed by a Tukey pairwise multiple-comparison test. The data represent averages SEM from at least five independent experiments. Statistical significance is indicated. A P value of 0.05 is shown as nonsignificant (n.s.).
Source publication
Nipah and Hendra viruses (NiV and HeV) exhibit high lethality in humans and are biosafety level 4 (BSL-4) paramyxoviruses in the growing genus Henipavirus. The attachment (G) and fusion (F) envelope glycoproteins are both required for viral entry into cells and for cell-cell fusion, which is pathognomonic of henipaviral infections. Here, we compare...
Contexts in source publication
Context 1
... tag insertions at the extracellular domains of the NiV and HeV F fusion proteins do not alter their fusogenic capacities. The NiV F (NF) and HeV F (HF) glycoproteins are class I viral fusion proteins with an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular domain (Fig. 1A). Intracellular . F 1 and F 2 subunits are held together by a disulfide bond. A FLAG tag was inserted just N terminal to the cathepsin L cleavage site (Cat L) located at the C terminus of F 2 , an extracellular portion of the F head domain. An AU1 tag, inserted at the intracellular C terminus of F (type I transmembrane protein), has ...
Context 2
... of Virology July 2019 Volume 93 Issue 13 e00577-19 jvi.asm.org 3 F 1 C-terminal tags (AU1) have been widely used in previous studies to detect henipavirus F glycoproteins and have not been shown to affect the functions of these proteins ( Fig. 1A) (8,10,19). However, henipaviral F proteins with identical extracellular tags that allow quantitative comparisons of cell surface expression (CSE) and fusion levels have not been described. Such constructs are necessary because cell-cell fusion is highly dependent on glycoprotein CSE (11). Although polyclonal antisera against NiV F have ...
Context 3
... insertion of a FLAG tag in the F ectodomain of the related morbilliviruses canine distemper virus (CDV) and measles virus (MeV) does not significantly modulate their bioactivity (40,41). In those studies, the FLAG tag was inserted in the F 2 subunit close to the furin cleavage site (Fig. 1A). The henipaviral Fs are processed by cathepsin L and not furin, and the sequences surrounding the protease cleavage site are very conserved among henipaviruses but much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit ...
Context 4
... In those studies, the FLAG tag was inserted in the F 2 subunit close to the furin cleavage site (Fig. 1A). The henipaviral Fs are processed by cathepsin L and not furin, and the sequences surrounding the protease cleavage site are very conserved among henipaviruses but much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit would alter the bioactivity of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or ...
Context 5
... much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit would alter the bioactivity of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or AU1)-or double (FLAG plus AU1)-tagged F constructs in combination with HA-tagged NiV G (NG) or HeV G (HG) constructs (Fig. 1B). HEK293T cells were transfected with F and G expression plasmids, and fusion levels were determined 16 to 20 h posttransfection (hpt) by counting ...
Context 6
... of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or AU1)-or double (FLAG plus AU1)-tagged F constructs in combination with HA-tagged NiV G (NG) or HeV G (HG) constructs (Fig. 1B). HEK293T cells were transfected with F and G expression plasmids, and fusion levels were determined 16 to 20 h posttransfection (hpt) by counting nuclei inside syncytia per field (200 field; five fields for each combination, n 4 independent experiments) and normalizing the fusion levels to that of the wild-type NiV fusion (FLAG NF and ...
Context 7
... levels to that of the wild-type NiV fusion (FLAG NF and NG, set to 100%). Neither the sequence, position of the tag, nor the presence of both tags (FLAG plus AU1) significantly altered the fusogenic capacity of the fusion protein significantly (P 0.05 by one-way analysis of variance [ANOVA] followed by a Tukey pairwise multiplecomparison test) (Fig. 1B). Therefore, we continued utilizing FLAG-tagged F constructs in subsequent experiments unless otherwise noted to allow us to quantify and compare henipaviral F glycoprotein cell surface expression ...
Context 8
... while homologous NiV and HeV F and G pairs yielded similar levels of cell-cell fusion, we observed fusion levels significantly lower than those of the wild type for the NF/HG pair (23.7%; P 0.0001) and greater than wild-type fusion levels for the HF/NG pair (150.2%; P 0.001) (Fig. ...
Context 9
... tag insertions at the extracellular domains of the NiV and HeV F fusion proteins do not alter their fusogenic capacities. The NiV F (NF) and HeV F (HF) glycoproteins are class I viral fusion proteins with an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular domain (Fig. 1A). Intracellular . F 1 and F 2 subunits are held together by a disulfide bond. A FLAG tag was inserted just N terminal to the cathepsin L cleavage site (Cat L) located at the C terminus of F 2 , an extracellular portion of the F head domain. An AU1 tag, inserted at the intracellular C terminus of F (type I transmembrane protein), has ...
Context 10
... of Virology July 2019 Volume 93 Issue 13 e00577-19 jvi.asm.org 3 F 1 C-terminal tags (AU1) have been widely used in previous studies to detect henipavirus F glycoproteins and have not been shown to affect the functions of these proteins ( Fig. 1A) (8,10,19). However, henipaviral F proteins with identical extracellular tags that allow quantitative comparisons of cell surface expression (CSE) and fusion levels have not been described. Such constructs are necessary because cell-cell fusion is highly dependent on glycoprotein CSE (11). Although polyclonal antisera against NiV F have ...
Context 11
... insertion of a FLAG tag in the F ectodomain of the related morbilliviruses canine distemper virus (CDV) and measles virus (MeV) does not significantly modulate their bioactivity (40,41). In those studies, the FLAG tag was inserted in the F 2 subunit close to the furin cleavage site (Fig. 1A). The henipaviral Fs are processed by cathepsin L and not furin, and the sequences surrounding the protease cleavage site are very conserved among henipaviruses but much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit ...
Context 12
... In those studies, the FLAG tag was inserted in the F 2 subunit close to the furin cleavage site (Fig. 1A). The henipaviral Fs are processed by cathepsin L and not furin, and the sequences surrounding the protease cleavage site are very conserved among henipaviruses but much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit would alter the bioactivity of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or ...
Context 13
... much less conserved between henipaviruses and morbilliviruses (i.e., CDV F) (Fig. 1A). To test whether an insertion of a FLAG tag near the C terminus of the F 2 subunit would alter the bioactivity of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or AU1)-or double (FLAG plus AU1)-tagged F constructs in combination with HA-tagged NiV G (NG) or HeV G (HG) constructs (Fig. 1B). HEK293T cells were transfected with F and G expression plasmids, and fusion levels were determined 16 to 20 h posttransfection (hpt) by counting ...
Context 14
... of NF or HF, we constructed NiV and HeV Fs that harbor either a C-terminal intracellular AU1 tag, an extracellular FLAG tag, or both (FLAG plus AU1) (Fig. 1A). We then compared the fusion capacities of the single (FLAG or AU1)-or double (FLAG plus AU1)-tagged F constructs in combination with HA-tagged NiV G (NG) or HeV G (HG) constructs (Fig. 1B). HEK293T cells were transfected with F and G expression plasmids, and fusion levels were determined 16 to 20 h posttransfection (hpt) by counting nuclei inside syncytia per field (200 field; five fields for each combination, n 4 independent experiments) and normalizing the fusion levels to that of the wild-type NiV fusion (FLAG NF and ...
Context 15
... levels to that of the wild-type NiV fusion (FLAG NF and NG, set to 100%). Neither the sequence, position of the tag, nor the presence of both tags (FLAG plus AU1) significantly altered the fusogenic capacity of the fusion protein significantly (P 0.05 by one-way analysis of variance [ANOVA] followed by a Tukey pairwise multiplecomparison test) (Fig. 1B). Therefore, we continued utilizing FLAG-tagged F constructs in subsequent experiments unless otherwise noted to allow us to quantify and compare henipaviral F glycoprotein cell surface expression ...
Citations
... Expression of singular viral proteins from plasmid-based expression systems in mammalian cell culture/organoids, the creation of BSL2 pseudotyped viruses that incorporate henipaviral glycoproteins, and the development of reverse genetics systems have enabled researchers to learn more about henipavirus entry, replication, and egress while also informing the development of countermeasures. Recently, the discovery of apathogenic henipaviruses (such as CedV) to study in lower containment settings have provided insights into henipaviral molecular pathogenesis in concert with findings from NiV and HeV infection experiments performed at BSL4 and clinical and laboratory findings from viral outbreaks [18,[46][47][48][49][50][51][52]. With the increasing diversity and number of relatively less pathogenic henipavirus isolates than NiV or HeV (such as CedV, LayV, and GAKV), researchers will have access to an unprecedented array of genetic models to safely investigate henipaviruses in low containment settings [3,5,6,8,9,11,33,41]. ...
Paramyxoviruses are a family of single-stranded negative-sense RNA viruses, many of which are responsible for a range of respiratory and neurological diseases in humans and animals. Among the most notable are the henipaviruses, which include the deadly Nipah (NiV) and Hendra (HeV) viruses, the causative agents of outbreaks of severe disease and high case fatality rates in humans and animals. NiV and HeV are maintained in fruit bat reservoirs primarily in the family Pteropus and spillover into humans directly or by an intermediate amplifying host such as swine or horses. Recently, non-chiropteran associated Langya (LayV), Gamak (GAKV), and Mojiang (MojV) viruses have been discovered with confirmed or suspected ability to cause disease in humans or animals. These viruses are less genetically related to HeV and NiV yet share many features with their better-known counterparts. Recent advances in surveillance of wild animal reservoir viruses have revealed a high number of henipaviral genome sequences distributed across most continents, and mammalian orders previously unknown to harbour henipaviruses. In this review, we summarize the current knowledge on the range of pathogenesis observed for the henipaviruses as well as their replication cycle, epidemiology, genomics, and host responses. We focus on the most pathogenic viruses, including NiV, HeV, LayV, and GAKV, as well as the experimentally non-pathogenic CedV. We also highlight the emerging threats posed by these and potentially other closely related viruses.
... However, there is no similar assay for measuring fusogenicity. Conventional approaches for quantifying fusogenicity often rely on split fluorescent protein systems [35][36][37][38][39][40] , such as the split GFP system that consists of GFP 1-10 and GFP 11 41 . For example, when cells that express hACE2 and GFP 1-10 are mixed with cells expressing SARS-CoV-2 S and GFP 11 , fusion occurs, and the resultant syncytia fluoresce green. ...
Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we establish a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrates a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, we apply this method to a region in the S2 domain that includes the first heptad repeat and central helix. Our results reveal that besides K986P and V987P, several mutations simultaneously improve expression and significantly lower the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.
... Previous reports have shown that recombinant NiV-G exhibited epitopes and structure necessary for specific antigen-antibody recognition (22). Since NiV and HeV glycoproteins are highly conserved and share 80% amino acid identity (23), and positive sera from NiV and HeV inoculated animals cross-reacts with NiV-G (22), so recombinant NiV-G is an ideal antigen for immunoassay. As expected the NiV-and HeV-positive sera showed reactivity to the recombinant NiV-G. ...
Introduction
Nipah virus (NiV) and Hendra virus (HeV), of the genus Henipavirus, family Paramyxoviridae, are classified as Risk Group 4 (RG4) pathogens that cause respiratory disease in pigs and acute/febrile encephalitis in humans with high mortality.
Methods
A competitive enzyme–linked immunosorbent assay (cELISA) using a monoclonal antibody (mAb) and recombinant NiV glycoprotein (G) was developed and laboratory evaluated using sera from experimental pigs, mini pigs and nonhuman primates. The test depends on competition between specific antibodies in positive sera and a virus–specific mAb for binding to NiV–G.
Results
Based on 1,199 negative and 71 NiV positive serum test results, the cutoff value was determined as 35% inhibition. The diagnostic sensitivity and specificity of the NiV cELISA was 98.58 and 99.92%, respectively. When testing sera from animals experimentally infected with NiV Malaysia, the cELISA detected antibodies from 14 days post–infection (dpi) and remained positive until the end of the experiment (28 dpi). Comparisons using the Kappa coefficient showed strong agreement (100%) between the cELISA and a plaque reduction neutralization test (PRNT).
Discussion
Because our cELISA is simpler, faster, and gives comparable or better results than PRNT, it would be an adequate screening test for suspect NiV and HeV cases, and it would also be useful for epidemiological surveillance of Henipavirus infections in different animal species without changing reagents.
... It has been statistically found out that the mortality rate of this viral disease is about 40-75%. In the case of Nipah virus survivors, it has been witnessed that some long-term side effects persisted in them like convulsions and certain changes in personality and certain other neurological disorders [26]. ...
... However, if vaccination of cattle is done at a reasonable price, it may appear to be successful in these areas. Despite these trends, pharmaceutical agencies are reluctant to invest in vaccine development courses such as against NiV, which are rare, despite high mortality [26]. ...
Viral diseases are causing mayhem throughout the world. One of the zoonotic viruses that have emerged as a potent threat to community health in the past few decades is Nipah virus. Nipah viral sickness is a zoonotic disease whose main carrier is bat. This disease is caused by Nipah virus (NiV). It belongs to the henipavirous group and of the family paramyxoviridae. Predominantly Pteropus spp. is the carrier of this virus. It was first reported from the Kampung Sungai Nipah town of Malaysia in 1998. Human-to-human transmission can also occur. Several repeated outbreaks were reported from South and Southeast Asia in the recent past. In humans, the disease is responsible for rapid development of acute illness, which can result in severe respiratory illness and serious encephalitis. Therefore, this calls for an urgent need for health authorities to conduct clinical trials to establish possible treatment regimens to prevent any further outbreaks.
... 4 The EFN proteins are normally bidirectional signaling proteins. 2,5 The EFNB2 and B3 are reported as the potential receptors interacting with the henipavirus glycoprotein for mediating the viral interaction, followed by the activation of fusion protein for membrane fusion and replication. 2,5 The henipavirus glycoprotein is a type II membrane protein that consists of a globular head, an N-terminus tail, a transmembrane domain, and a stalk domain; however, the EFN receptors are bound to the central cavity of the b-propeller head domain of both the NiV and HeV glycoproteins. ...
... 2,5 The EFNB2 and B3 are reported as the potential receptors interacting with the henipavirus glycoprotein for mediating the viral interaction, followed by the activation of fusion protein for membrane fusion and replication. 2,5 The henipavirus glycoprotein is a type II membrane protein that consists of a globular head, an N-terminus tail, a transmembrane domain, and a stalk domain; however, the EFN receptors are bound to the central cavity of the b-propeller head domain of both the NiV and HeV glycoproteins. 4,5 Molecular characterization and various computer-aided drug discovery methods have been proposed to identify novel inhibitors to combat the NiV and HeV viral entry into the human cell. ...
... 2,5 The henipavirus glycoprotein is a type II membrane protein that consists of a globular head, an N-terminus tail, a transmembrane domain, and a stalk domain; however, the EFN receptors are bound to the central cavity of the b-propeller head domain of both the NiV and HeV glycoproteins. 4,5 Molecular characterization and various computer-aided drug discovery methods have been proposed to identify novel inhibitors to combat the NiV and HeV viral entry into the human cell. 6,7 Furthermore, various neutralizing vaccines have been developed and reported to bind to the human EFN G-H loop to disrupt the attachment of Henipavirus glycoprotein followed by deactivating the fusion protein thereby stopping the membrane fusion and replication of the virus in the human cell. ...
Unlabelled:
Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic paramyxovirus which belongs to Henipavirus family, causes severe respiratory disease, and may lead to fatal encephalitis infections in humans. NiV and HeV glycoproteins (G) bind to the highly conserved human ephrin-B2 and B3 (EFNB2 & EFNB3) cell surface proteins to mediate the viral entry. In this study, various molecular modelling approaches were employed to understand protein-protein interaction (PPI) of NiV and HeV glycoprotein (84% sequence similarity) with Human EFN (B2 and B3) to investigate the molecular mechanism of interaction at atomic level. Our computational study emphasized the PPI profile of both the viral glycoproteins with EFN (B2 and B3) in terms of non-bonded contacts, H-bonds, salt bridges, and identification of interface hotspot residues which play a critical role in the formation of complexes that mediate viral fusion and entry into the host cell. According to the reports, EFNB2 is considered to be more actively involved in the attachment with the NiV and HeV glycoprotein; interestingly the current computational study has displayed more conformational stability in HeV/NiV glycoprotein with EFNB2 complex with relatively high binding energy as compared to EFNB3. During the MD simulation, the number of H-bond formations was observed to be less in the case of EFNB3 complexes, which may be the possible reason for less conformational stability in the EFNB3 complexes. The current detailed interaction study on the PPI may put a path forward in designing peptide inhibitors to obstruct the interaction of viral glycoproteins with host proteins, thereby inhibiting viral entry.
Graphical abstract:
The viral attachment and fusion of Nipah and Hendra virus was explored through the interaction between viral glycoprotein and the host cell surface ephrin protein. The MD simulation results displayed more stability in Nipah and Hendra glycoprotein with EFNB2 as compared to EFNB3. The residue Glu533 in the central cavity of HeV/NiV glycoprotein protein identified as the potential hotspot in binding with the G-H loop of EFNB2.
Supplementary information:
The online version contains supplementary material available at 10.1007/s12039-022-02110-9.
... Interestingly, the levels of incorporation of the cysteine proteins were generally lower than those for wild-type NiV F and G, which may account for some of the lack of viral entry for some of the cysteine mutants (Fig. 5B). The findings that the levels of viral entry and syncytium formation are not always equivalent has been previously observed (31,35,36,46,47). We propose that the S179C mutant can effectively promote the formation of fusion pores sufficiently large to allow protein mixing (in our DSP assay), as well as viral genome entry into cells, but not extensive enough to allow nuclei movement and gathering during the process of cell-cell fusion. ...
... Importantly, these tagged proteins have been shown to be fully functional (29,30). The surface glycoproteins were then detected from the total cell lysates or the immunoprecipitated eluate via immunoblotting using either rabbit anti-HA (NiV G) or mouse anti-AU1 (NiV F) (Fig. 6A) antibodies, as we previously established (35,47). Interestingly, we observed a significant 4-to 9-fold increase in the avidities of G/F interactions for the hypofusogenic G stalk mutants, while the A86C mutant, which yielded near-wild-type levels of syncytium formation, also yielded a near-wild-type avidity of G/F interactions (Fig. 6B). ...
... The coimmunoprecipitation assays revealed that most cysteine mutants were capable of interacting with NiV F at avidity levels significantly higher than those of the wild type. Typically for the henipaviruses, the binding avidity of G and F mutants inversely correlates with their respective levels of fusogenicity, as we previously reported (29)(30)(31)47). These inverse correlations support the G/F dissociation models, whereby the interactions between G and F must decrease so the membrane fusion cascade may proceed. ...
The important Paramyxoviridae family includes measles, mumps, human parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). The deadly emerging NiV can cause neurologic and respiratory symptoms in humans with a >60% mortality rate.
... When HP binds to the HS receptors on host cells, there is no or limited ability for Hendra and Nipah to show trans infection between host cells [19] Hendra and Nipah are in the same family and genus but are distinctly different viruses (Figure 4). Although their glycoproteins seem to react similarly when treated with antibodies specific to the fusion protein, it should be noted that these studies all use anti-Hendra fusion protein antibodies against Hendra and antibodies specific to the fusion protein against Nipah [100]. Researchers discovered that the G protein is more important to Nipah infection than its fusion protein, whereas the fusion protein in Hendra is the main component of membrane attachment [100]. ...
... Although their glycoproteins seem to react similarly when treated with antibodies specific to the fusion protein, it should be noted that these studies all use anti-Hendra fusion protein antibodies against Hendra and antibodies specific to the fusion protein against Nipah [100]. Researchers discovered that the G protein is more important to Nipah infection than its fusion protein, whereas the fusion protein in Hendra is the main component of membrane attachment [100]. Molecular docking studies have been used to find common peptides that may inhibit the binding of glycoproteins on the surface of the Nipah virus [101]. ...
... against Nipah [100]. Researchers discovered that the G protein is more important to Nipah infection than its fusion protein, whereas the fusion protein in Hendra is the main component of membrane attachment [100]. ...
Zoonotic diseases are infectious diseases that pass from animals to humans. These include diseases caused by viruses, bacteria, fungi, and parasites and can be transmitted through close contact or through an intermediate insect vector. Many of the world’s most problematic zoonotic diseases are viral diseases originating from animal spillovers. The Spanish influenza pandemic, Ebola outbreaks in Africa, and the current SARS-CoV-2 pandemic are thought to have started with humans interacting closely with infected animals. As the human population grows and encroaches on more and more natural habitats, these incidents will only increase in frequency. Because of this trend, new treatments and prevention strategies are being explored. Glycosaminoglycans (GAGs) are complex linear polysaccharides that are ubiquitously present on the surfaces of most human and animal cells. In many infectious diseases, the interactions between GAGs and zoonotic pathogens correspond to the first contact that results in the infection of host cells. In recent years, researchers have made progress in understanding the extraordinary roles of GAGs in the pathogenesis of zoonotic diseases, suggesting potential therapeutic avenues for using GAGs in the treatment of these diseases. This review examines the role of GAGs in the progression, prevention, and treatment of different zoonotic diseases caused by viruses.
... The relative levels of syncytia formation across HNVs remain understudied. We recently reported that NiV and HeV induce similar cell-cell fusion and viral entry levels (20), likely attributed to their G/F high sequence similarities. However, NiV and CedV G/ F have significantly lower amino acid sequence identities (Fig. 1A), which may cause different fusogenic capacities, which we explored in this study. ...
... Cedar virus is significantly less fusogenic than Nipah virus. To accurately quantify and compare fusogenicity (cell-cell fusion) and cell surface expression (CSE) levels between NiV and CedV, we introduced extracellular hemagglutinin (HA) and FLAG tags into codon-optimized CedV G and F, respectively, to match the functional NiV G and F constructs (17,18,20). Then, since cell-cell fusion levels depend on CSE levels (26), we optimized transfections of NiV G/NiV F and CedV G/CedV F plasmid pairs into HEK 293T cells to obtain comparable CSE levels. ...
... Then, since cell-cell fusion levels depend on CSE levels (26), we optimized transfections of NiV G/NiV F and CedV G/CedV F plasmid pairs into HEK 293T cells to obtain comparable CSE levels. We then quantified the number of nuclei inside syncytia per area 20 h posttransfection (hpt), as previously performed (17,18,20,27). As the CSE levels of HNV glycoproteins affect their fusogenicity levels (20), to accurately compare NiV and CedV intrinsic fusogenic G/F capacities, we calculated fusion indices (FI), defined as the ratio of cell-cell fusion levels to G or F CSE levels. ...
Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity.
IMPORTANCE
The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.
... Yet, there remains a lack of empirical knowledge on the contribution of direct cell-to-cell transmission on viral propagation and disease development. Understanding the contribution of syncytium formation to pathogenesis would also inform on whether in vitro cell-cell fusion assays, which are widely used to functionally describe viruses [20][21][22][23][24], are useful predictors of in vivo dynamics. ...
... Furthermore, N-and O-glycans in most paramyxoviral glycoproteins have been shown to modulate protein expression, folding, and transport; for HNVs in particular, several N-and O-glycans in NiV and HeV G and F modulate virus-cell and cell-cell fusion independently of protein expression [64][65][66]. It is worth noting that, although membrane fusion levels induced by NiV and HeV are relatively similar [24], some N-and O-glycans of NiV and HeV G and F modulate syncytium formation differently [67][68][69]. In addition, while cell-cell fusion and virus-cell fusion mechanisms are presumably driven by similar underlying mechanisms, incongruities have been reported. ...
... Comparing intensities of syncytium formation between in vitro and in vivo conditions is thus challenging because the methods used for syncytium quantification are different, but also because the definition of syncytium differs. Some in vivo studies mention syncytia with 2 nuclei (e.g., [75]), while many in vitro studies using manual counting of syncytia (in opposition to reporter genes) only consider cells with four or more nuclei (e.g., [24,69]) as rare small nuclei can be observed in cell cultures independently of the presence of viral proteins. ...
Syncytium formation, i.e., cell–cell fusion resulting in the formation of multinucleated cells, is a hallmark of infection by paramyxoviruses and other pathogenic viruses. This natural mechanism has historically been a diagnostic marker for paramyxovirus infection in vivo and is now widely used for the study of virus-induced membrane fusion in vitro. However, the role of syncytium formation in within-host dissemination and pathogenicity of viruses remains poorly understood. The diversity of henipaviruses and their wide host range and tissue tropism make them particularly appropriate models with which to characterize the drivers of syncytium formation and the implications for virus fitness and pathogenicity. Based on the henipavirus literature, we summarized current knowledge on the mechanisms driving syncytium formation, mostly acquired from in vitro studies, and on the in vivo distribution of syncytia. While these data suggest that syncytium formation widely occurs across henipaviruses, hosts, and tissues, we identified important data gaps that undermined our understanding of the role of syncytium formation in virus pathogenesis. Based on these observations, we propose solutions of varying complexity to fill these data gaps, from better practices in data archiving and publication for in vivo studies, to experimental approaches in vitro.
... Yet, there remains a lack of empirical knowledge on the contribution of direct cell-to-cell transmission on viral propagation and disease development. Understanding the contribution of syncytium formation to pathogenesis would also inform on whether in vitro cell-cell fusion assays, which are widely used to functionally describe viruses [17][18][19][20][21], are useful predictors of in vivo dynamics. ...
... Finally, N-and O-glycans in most paramyxoviral glycoproteins have been shown to modulate protein expression, folding and transport; for HNVs in particular, several N-and O-glycans in NiV and HeV G and F modulate virus-cell and cell-cell fusion independently of protein expression [60][61][62]. It is worth noting that, although membrane fusion levels induced by NiV and HeV are similar [21], some N-and O-glycans of NiV and HeV G and F modulate syncytium formation differently [63][64][65]. In addition, while cell-cell fusion and virus-cell fusion mechanisms are presumably driven by similar underlying mechanisms, incongruities have been reported. ...
Syncytium formation, i.e., cell-cell fusion resulting in the formation of multinucleated cells, is a hallmark of infection by paramyxoviruses and other important viruses. This natural mechanism has historically been a diagnostic marker for paramyxovirus infection in vivo and is now widely studied for virus-induced membrane fusion in vitro. However, the role of syncytium formation in within-host dissemination and pathogenicity of viruses remains poorly understood. The diversity of henipaviruses and their wide host range and tissue tropism make them particularly appropriate models to characterize the drivers of syncytium formation and its implications for virus fitness and pathogenicity. Based on the henipavirus literature, we summarized current knowledge on the mechanisms driving syncytium formation, mostly acquired from in vitro studies, and on the in vivo distribution of syncytia. While these data suggest that syncytium formation widely occurs across henipaviruses, hosts and tissues, we identified important data gaps that undermined our understanding of the role of syncytium formation in virus pathogenesis. Based on these observations, we propose solutions of varying complexity to fill these data gaps, from better practices in data archiving and publication for in vivo studies, to experimental approaches in vitro.
