Potent human neutralizing antibodies against Nipah virus derived from two ancestral antibody heavy chains.

Nipah virus (NiV) is a World Health Organization priority pathogen and there are currently no approved drugs for clinical immunotherapy. Through the use of a naïve human phage-displayed Fab library, two neutralizing antibodies (NiV41 and NiV42) targeting the NiV receptor binding protein (RBP) were identified. Following affinity maturation, antibodies derived from NiV41 display cross-reactivity against both NiV and Hendra virus (HeV), whereas the antibody based on NiV42 is only specific to NiV. Results of immunogenetic analysis reveal a correlation between the maturation of antibodies and their antiviral activity. In vivo testing of NiV41 and its mature form (41-6) show protective efficacy against a lethal NiV challenge in hamsters. Furthermore, a 2.88 Å Cryo-EM structure of the tetrameric RBP and antibody complex demonstrates that 41-6 blocks the receptor binding interface. These findings can be beneficial for the development of antiviral drugs and the design of vaccines with broad spectrum against henipaviruses.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Prefusion stabilization of the Hendra and Langya virus F proteins.

Nipah virus (NiV) and Hendra virus (HeV) are pathogenic paramyxoviruses that cause mild-to-severe disease in humans. As members of the Henipavirus genus, NiV and HeV use an attachment (G) glycoprotein and a class I fusion (F) glycoprotein to invade host cells. The F protein rearranges from a metastable prefusion form to an extended postfusion form to facilitate host cell entry. Prefusion NiV F elicits higher neutralizing antibody titers than postfusion NiV F, indicating that stabilization of prefusion F may aid vaccine development. A combination of amino acid substitutions (L104C/I114C, L172F, and S191P) is known to stabilize NiV F in its prefusion conformation, although the extent to which substitutions transfer to other henipavirus F proteins is not known. Here, we perform biophysical and structural studies to investigate the mechanism of prefusion stabilization in F proteins from three henipaviruses: NiV, HeV, and Langya virus (LayV). Three known stabilizing substitutions from NiV F transfer to HeV F and exert similar structural and functional effects. One engineered disulfide bond, located near the fusion peptide, is sufficient to stabilize the prefusion conformations of both HeV F and LayV F. Although LayV F shares low overall sequence identity with NiV F and HeV F, the region around the fusion peptide exhibits high sequence conservation across all henipaviruses. Our findings indicate that substitutions targeting this site of conformational change might be applicable to prefusion stabilization of other henipavirus F proteins and support the use of NiV as a prototypical pathogen for henipavirus vaccine antigen design.IMPORTANCEPathogenic henipaviruses such as Nipah virus (NiV) and Hendra virus (HeV) cause respiratory symptoms, with severe cases resulting in encephalitis, seizures, and coma. The work described here shows that the NiV and HeV fusion (F) proteins share common structural features with the F protein from an emerging henipavirus, Langya virus (LayV). Sequence alignment alone was sufficient to predict which known prefusion-stabilizing amino acid substitutions from NiV F would stabilize the prefusion conformations of HeV F and LayV F. This work also reveals an unexpected oligomeric interface shared by prefusion HeV F and NiV F. Together, these advances lay a foundation for future antigen design targeting henipavirus F proteins. In this way, Nipah virus can serve as a prototypical pathogen for the development of protective vaccines and monoclonal antibodies to prepare for potential henipavirus outbreaks.

The cryo-EM structure of homotetrameric attachment glycoprotein from langya henipavirus.

Langya Henipavirus (LayV) infection is an emerging zoonotic disease that has been causing respiratory symptoms in China since 2019. For virus entry, LayV's genome encodes the fusion protein F and the attachment glycoprotein G. However, the structural and functional information regarding LayV-G remains unclear. In this study, we revealed that LayV-G cannot bind to the receptors found in other HNVs, such as ephrin B2/B3, and it shows different antigenicity from HeV-G and NiV-G. Furthermore, we determined the near full-length structure of LayV-G, which displays a distinct mushroom-shaped configuration, distinguishing it from other attachment glycoproteins of HNV. The stalk and transmembrane regions resemble the stem and root of mushroom and four downward-tilted head domains as mushroom cap potentially interact with the F protein and influence membrane fusion process. Our findings enhance the understanding of emerging HNVs that cause human diseases through zoonotic transmission and provide implication for LayV related vaccine development.

Bioinformatic investigation of Nipah virus surface protein mutations: Molecular docking with Ephrin B2 receptor, molecular dynamics simulation, and structural impact analysis.

The SARS-CoV-2 outbreak resulted in significant challenges and loss of life. The Nipah virus, known for its high infectivity and severity, was designated an emergency concern by the World Health Organization. To understand its mutations, the Nipah virus proteins were analyzed extensively, with a focus on the essential G and F proteins responsible for viral entry into host cells. Our bioinformatics analysis unveiled multiple mutations, including simultaneous mutations within a single sequence. Notably, the G273S mutation in the F protein was identified as a potential cause of structural damage, which carries significant implications for vaccine development. Comparing the docking scores of G and F proteins with the Ephrin B2 receptor, it was found that the Y228H mutation in the G protein and the D252G mutation in the F protein likely affect virus entry into host cells. Moreover, our investigation into stability and deformability highlighted the impact of the Y228H mutation in the G protein complex. Molecular dynamics simulations revealed increased flexibility and conformational changes in the G protein complex with the Y228H mutation compared with the known complex. Furthermore, evaluating the root mean square deviation variation demonstrated greater dynamic behavior in the G protein complex and the Ephrin B2 receptor complex. This comprehensive study provides valuable insights into Nipah virus mutations, their significance for vaccine development, and the importance of understanding protein complex behavior in drug discovery. The identified mutations, especially G273S and Y228H, hold crucial implications for future research and potential interventions against the Nipah virus.

Nipah virus attachment glycoprotein ectodomain delivered by type 5 adenovirus vector elicits broad immune response against NiV and HeV.

Nipah virus (NiV) and Hendra virus (HeV) are newly emerging dangerous zoonotic pathogens of the Henipavirus genus of the Paramyxoviridae family. NiV and HeV (HNVs) which are transmitted by bats cause acute respiratory disease and fatal encephalitis in humans. To date, as there is a lack of antiviral drugs or effective antiviral therapies, the development of vaccines against those two viruses is of primary importance, and the immunogen design is crucial to the success of vaccines. In this study, the full-length protein (G), the ectodomain (Ge) and the head domain (Gs) of NiV attachment glycoprotein were delivered by the replication-defective type 5 adenovirus vector (Ad5) respectively, and the recombinant Ad5-NiV vaccine candidates (Ad5-NiVG, Ad5-NiVGe and Ad5-NiVGs) were constructed and their immunogenicity were evaluated in mice. The results showed that all the vaccine candidates stimulated specific humoral and cellular immune responses efficiently and rapidly against both NiV and HeV, and the Ad5-NiVGe elicited the strongest immune responses after a single-dose immunization. Furthermore, the potent conserved T-cell epitope DTLYFPAVGFL shared by NiV and HeV was identified in the study, which may provide valid information on the mechanism of HNVs-specific cellular immunity. In summary, this study demonstrates that the Ad5-NiVGe could be a potent vaccine candidate against HNVs by inducing robust humoral and cellular immune responses.

Computer-assisted identification of potential quinolone derivatives targeting Nipah virus glycoprotein attachment with human cell surface receptor ephrin-B2: Multistep virtual screening.

Nipah Virus (NiV) is a single-stranded, negative-sense, highly lethal RNA virus. Even though NiV has close to 70-80% of mortality in India and Bangladesh, still there is no available US FDA-approved drug or vaccine. NiV attachment glycoprotein (NiV-G) is critical for NiV to invade the human cell where ephrinB2 which is a crucial membrane-bound ligand that acts as a target of NiV. Most of the research has been performed targeting NiV or human ephrin-B to date. Quinolone derivatives are proven scaffolds for many approved drugs used to treat various bacterial, viral respiratory tract, and urinary tract infections, and rheumatologic disorders such as systemic lupus erythematosus, rheumatoid arthritis. Therefore, we have tried to find potential drug molecules employing quinolone scaffold-based derivatives from PubChem targeting both NiV-G and ephrin-B2 protein. A total of 1500+ quinolone derivatives were obtained from PubChem which were screened based on Drug Likeness followed by being subjected to XP docking employing Schrödinger software. The top ten best molecules were then chosen for their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling based on the docking score ranking. Further, the top five molecules were selected for 200ns molecular dynamics (MD) simulation study with Desmond module followed by MM-GBSA study by Prime module of Schrödinger. The exhaustive analysis leads us to the top three probable lead drug molecules for NiV are PubChem CID 23646770, an analog of PubChem CID 67726448, and PubChem CID 10613168 which have predicted Ki values of 0.480 µm, 0.785 µm, and 0.380 µm, respectively. These proposed molecules can be the future drugs targeting NiV-G and human ephrin-B2 which requires further in vivo validation.

Structure and antigenicity of divergent Henipavirus fusion glycoproteins.

In August 2022, a novel henipavirus (HNV) named Langya virus (LayV) was isolated from patients with severe pneumonic disease in China. This virus is closely related to Mòjiang virus (MojV), and both are divergent from the bat-borne HNV members, Nipah (NiV) and Hendra (HeV) viruses. The spillover of LayV is the first instance of a HNV zoonosis to humans outside of NiV and HeV, highlighting the continuing threat this genus poses to human health. In this work, we determine the prefusion structures of MojV and LayV F proteins via cryogenic electron microscopy to 2.66 and 3.37 Å, respectively. We show that despite sequence divergence from NiV, the F proteins adopt an overall similar structure but are antigenically distinct as they do not react to known antibodies or sera. Glycoproteomic analysis revealed that while LayV F is less glycosylated than NiV F, it contains a glycan that shields a site of vulnerability previously identified for NiV. These findings explain the distinct antigenic profile of LayV and MojV F, despite the extent to which they are otherwise structurally similar to NiV. Our results carry implications for broad-spectrum HNV vaccines and therapeutics, and indicate an antigenic, yet not structural, divergence from prototypical HNVs.

Experimental infection of tick cells with Nipah virus.

Nipah virus (NiV), a highly pathogenic henipavirus of the family Paramyxoviridae, which causes fatal encephalitis in 40-70% of affected patients, was first reported in Malaysia over 20 years ago. Pteropid bats are the natural hosts of henipaviruses, and ticks have been proposed as a possible link between bats and mammalian hosts. To investigate this hypothesis, infection of the tick cell line IDE8 with NiV was examined. Presence of viral RNA and antigen in the NiV-infected tick cells was confirmed. Infectious virions were recovered from NiV-infected tick cells and ultrastructural features of NiV were observed by electron microscopy. These results suggest that ticks could support NiV infection, potentially playing a role in transmission.

Structural basis for antibody recognition of vulnerable epitopes on Nipah virus F protein.

Nipah virus (NiV) is a pathogenic paramyxovirus that causes fatal encephalitis in humans. Two envelope glycoproteins, the attachment protein (G/RBP) and fusion protein (F), facilitate entry into host cells. Due to its vital role, NiV F presents an attractive target for developing vaccines and therapeutics. Several neutralization-sensitive epitopes on the NiV F apex have been described, however the antigenicity of most of the F protein's surface remains uncharacterized. Here, we immunize mice with prefusion-stabilized NiV F and isolate ten monoclonal antibodies that neutralize pseudotyped virus. Cryo-electron microscopy reveals eight neutralization-sensitive epitopes on NiV F, four of which have not previously been described. Novel sites span the lateral and basal faces of NiV F, expanding the known library of vulnerable epitopes. Seven of ten antibodies bind the Hendra virus (HeV) F protein. Multiple sequence alignment suggests that some of these newly identified neutralizing antibodies may also bind F proteins across the Henipavirus genus. This work identifies new epitopes as targets for therapeutics, provides a molecular basis for NiV neutralization, and lays a foundation for development of new cross-reactive antibodies targeting Henipavirus F proteins.

Pseudotyped Virus for Henipavirus.

The genus Henipavirus (HNV) includes two virulent infectious viruses, Nipah virus (NiV) and Hendra virus (HeV), which are the focus of considerable public health research efforts and have been classified as priority infectious diseases by the World Health Organization. Both viruses are high risk and should be handled in biosafety level 4 laboratories. Pseudotyped viruses containing the envelope proteins of HNV viruses have the same envelope protein structure as the authentic viruses; thus, they can mimic the receptor-binding and membrane fusion processes of authentic viruses with host cells and can be handled in biosafety level 2 laboratories. These characteristics enable pseudotyped viruses to be widely used in studies of viral infection mechanisms (packaging, budding, virus attachment, membrane fusion, viral entry, and glycosylation), inhibitory drug screening assays, and monoclonal antibody neutralization characteristics. This review will provide an overview of the progress of research concerning pseudotyped virus packaging systems for NiV and HeV.

Computational identification of drug-like marine natural products as potential RNA polymerase inhibitors against Nipah virus.

Nipah virus (NiV) has been an alarming threat to human populations in southern Asia for more than a decade. It is one of the most deadly viruses in the Mononegavirales order. Despite its high mortality rate and virulence, no chemotherapeutic agent or vaccine is publicly available. Hence, this work was conducted to computationally screen marine natural products database for drug-like potential inhibitors for the viral RNA-dependent RNA polymerase (RdRp). The structural model was subjected to molecular dynamics (MD) simulation to obtain the native ensemble of the protein. The CMNPDB dataset of marine natural products was filtered to retain only compounds following Lipinski's five rules. The molecules were energy minimized and docked into different conformers of the RdRp using AutoDock Vina. The best 35 molecules were rescored by GNINA, a deep learning-based docking software. The resulting nine compounds were evaluated for their pharmacokinetic profiles and medicinal chemistry properties. The best five compounds were subjected to MD simulation for 100 ns, followed by binding free energy estimation via Molecular Mechanics/ Generalized Born Surface Area (MM/GBSA) calculations. The results showed remarkable behavior of five hits as inferred by stable binding pose and orientation to block the exit channel of RNA synthesis products in the RdRp cavity. These hits are promising starting materials for in vitro validation and structural modifications to enhance the pharmacokinetic and medicinal chemistry properties for developing antiviral lead compounds.

Orthoparamyxovirinae C Proteins Have a Common Origin and a Common Structural Organization.

The protein C is a small viral protein encoded in an overlapping frame of the P gene in the subfamily Orthoparamyxovirinae. This protein, expressed by alternative translation initiation, is a virulence factor that regulates viral transcription, replication, and production of defective interfering RNA, interferes with the host-cell innate immunity systems and supports the assembly of viral particles and budding. We expressed and purified full-length and an N-terminally truncated C protein from Tupaia paramyxovirus (TupV) C protein (genus Narmovirus). We solved the crystal structure of the C-terminal part of TupV C protein at a resolution of 2.4 Å and found that it is structurally similar to Sendai virus C protein, suggesting that despite undetectable sequence conservation, these proteins are homologous. We characterized both truncated and full-length proteins by SEC-MALLS and SEC-SAXS and described their solution structures by ensemble models. We established a mini-replicon assay for the related Nipah virus (NiV) and showed that TupV C inhibited the expression of NiV minigenome in a concentration-dependent manner as efficiently as the NiV C protein. A previous study found that the Orthoparamyxovirinae C proteins form two clusters without detectable sequence similarity, raising the question of whether they were homologous or instead had originated independently. Since TupV C and SeV C are representatives of these two clusters, our discovery that they have a similar structure indicates that all Orthoparamyxovirine C proteins are homologous. Our results also imply that, strikingly, a STAT1-binding site is encoded by exactly the same RNA region of the P/C gene across Paramyxovirinae, but in different reading frames (P or C), depending on which cluster they belong to.

Molecular docking and dynamics studies for the identification of Nipah virus glycoprotein inhibitors from Indian medicinal plants.

The infection by Nipah Virus (NiV), a zoonotic paramyxovirus, is fatal and several outbreaks have been reported in humans in various countries. No effective vaccines or drugs are developed till date to control this infection. The NiV-Glycoprotein (NiV-G) is one of the essential proteins for viral entry by binding to the Ephrin-B receptors. The present study screens the potential phytocompounds that can target NiV-G and thereby inhibit the viral entry to human. Computer-aided virtual screening of 1426 phytocompounds from various medicinal plants was carried out to investigate their efficacy as potential therapeutics. Ribavirin, the currently used drug, was also docked to compare the docking score and intermolecular interactions between ligand and target protein. Further, molecular dynamics simulations and MM-PBSA binding free energy calculations were performed to understand the stability of the docked complexes. Radius of gyrations and Solvent Accessible Surface Area were also performed to evaluate the compactness and solvent behaviour of ligand-receptor complexes during the 100 ns simulation. Our analysis revealed that the alkaloid, Serpentinine, has the highest potency to block NiV-G with favourable binding.Communicated by Ramaswamy H. Sarma.

IFITM3 promotes NiV envelope protein-mediated entry into MDCK cells and interacts with the fusion subunit of the F protein.

IFITM proteins are a host restriction factor with broad-spectrum antiviral activity, but the role in the paramyxovirus entry remains unclear. Nipah virus (NiV) is a zoonotic virus of the paramyxoviridae with extremely high lethality. Here, we assessed the role of IFITM3 on NiV G and F glycoprotein-mediated virus entry. Using NiV pseudovirus bearing NiV G and F proteins to infect IFITM3-induced MDCK cells, we found that overexpression of IFITM3 promotes NiV G and F proteins-mediated virus entry. Mechanistically, the subcellular distribution showed that F protein completely co-localized with IFITM3, but G protein does not. Immunoprecipitation further indicated that IFITM3 strongly captures F protein rather than G protein. F protein truncation found that the F1 subunit completely co-localized and captures with IFITM3, but not the F2 subunit. Furthermore, IFITM3 strongly binds to F1 truncations containing fusion peptide (FP), and F1 strongly captures IFITM3 truncation with the intramembrane domain (IMD). Together, the results suggest that IFITM3 can promote NiV G and F proteins-mediated virus entry into MDCK cells, and IFITM3 directly interacts with the F1 subunit of NiV F protein dependent on the former's IMD and the latter's FP, which may occur after incorporation of fusion peptides into the cell membrane following virus fusion activation.

Neurological pathophysiology of SARS-CoV-2 and pandemic potential RNA viruses: a comparative analysis.

SARS-CoV-2 has infected hundreds of millions of people with over four million dead, resulting in one of the worst global pandemics in recent history. Neurological symptoms associated with COVID-19 include anosmia, ageusia, headaches, confusion, delirium, and strokes. These may manifest due to viral entry into the central nervous system (CNS) through the blood-brain barrier (BBB) by means of ill-defined mechanisms. Here, we summarize the abilities of SARS-CoV-2 and other neurotropic RNA viruses, including Zika virus and Nipah virus, to cross the BBB into the CNS, highlighting the role of magnetic resonance imaging (MRI) in assessing presence and severity of brain structural changes in COVID-19 patients. We present new insight into key mutations in SARS-CoV-2 variants B.1.1.7 (P681H) and B.1.617.2 (P681R), which may impact on neuropilin 1 (NRP1) binding and CNS invasion. We postulate that SARS-CoV-2 may infect both peripheral cells capable of crossing the BBB and brain endothelial cells to traverse the BBB and spread into the brain. COVID-19 patients can be followed up with MRI modalities to better understand the long-term effects of COVID-19 on the brain.

Viral Interactions with Adaptor-Protein Complexes: A Ubiquitous Trait among Viral Species.

Numerous viruses hijack cellular protein trafficking pathways to mediate cell entry or to rearrange membrane structures thereby promoting viral replication and antagonizing the immune response. Adaptor protein complexes (AP), which mediate protein sorting in endocytic and secretory transport pathways, are one of the conserved viral targets with many viruses possessing AP-interacting motifs. We present here different mechanisms of viral interference with AP complexes and the functional consequences that allow for efficient viral propagation and evasion of host immune defense. The ubiquity of this phenomenon is evidenced by the fact that there are representatives for AP interference in all major viral families, covered in this review. The best described examples are interactions of human immunodeficiency virus and human herpesviruses with AP complexes. Several other viruses, like Ebola, Nipah, and SARS-CoV-2, are pointed out as high priority disease-causative agents supporting the need for deeper understanding of virus-AP interplay which can be exploited in the design of novel antiviral therapies.

Roles of Cholesterol in Early and Late Steps of the Nipah Virus Membrane Fusion Cascade.

Cholesterol has been implicated in various viral life cycle steps for different enveloped viruses, including viral entry into host cells, cell-cell fusion, and viral budding from infected cells. Enveloped viruses acquire their membranes from their host cells. Although cholesterol has been associated with the binding and entry of various enveloped viruses into cells, cholesterol's exact function in the viral-cell membrane fusion process remains largely elusive, particularly for the paramyxoviruses. Furthermore, paramyxoviral fusion occurs at the host cell membrane and is essential for both virus entry (virus-cell fusion) and syncytium formation (cell-cell fusion), central to viral pathogenicity. Nipah virus (NiV) is a deadly member of the Paramyxoviridae family, which also includes Hendra, measles, mumps, human parainfluenza, and various veterinary viruses. The zoonotic NiV causes severe encephalitis, vasculopathy, and respiratory symptoms, leading to a high mortality rate in humans. We used NiV as a model to study the role of membrane cholesterol in paramyxoviral membrane fusion. We used a combination of methyl-beta cyclodextrin (MßCD), lovastatin, and cholesterol to deplete or enrich cell membrane cholesterol outside cytotoxic concentrations. We found that the levels of cellular membrane cholesterol directly correlated with the levels of cell-cell fusion induced. These phenotypes were paralleled using NiV/vesicular stomatitis virus (VSV)-pseudotyped viral infection assays. Remarkably, our mechanistic studies revealed that cholesterol reduces an early F-triggering step but enhances a late fusion pore formation step in the NiV membrane fusion cascade. Thus, our results expand our mechanistic understanding of the paramyxoviral/henipaviral entry and cell-cell fusion processes.IMPORTANCE Cholesterol has been implicated in various steps of the viral life cycle for different enveloped viruses. Nipah virus (NiV) is a highly pathogenic enveloped virus in the Henipavirus genus within the Paramyxoviridae family, capable of causing a high mortality rate in humans and high morbidity in domestic and agriculturally important animals. The role of cholesterol for NiV or the henipaviruses is unknown. Here, we show that the levels of cholesterol influence the levels of NiV-induced cell-cell membrane fusion during syncytium formation and virus-cell membrane fusion during viral entry. Furthermore, the specific role of cholesterol in membrane fusion is not well defined for the paramyxoviruses. We show that the levels of cholesterol affect an early F-triggering step and a late fusion pore formation step during the membrane fusion cascade. Thus, our results expand our mechanistic understanding of the viral entry and cell-cell fusion processes, which may aid the development of antivirals.

Predicting substitutions to modulate disorder and stability in coiled-coils.

BACKGROUND: Coiled-coils are described as stable structural motifs, where two or more helices wind around each other. However, coiled-coils are associated with local mobility and intrinsic disorder. Intrinsically disordered regions in proteins are characterized by lack of stable secondary and tertiary structure under physiological conditions in vitro. They are increasingly recognized as important for protein function. However, characterizing their behaviour in solution and determining precisely the extent of disorder of a protein region remains challenging, both experimentally and computationally. RESULTS: In this work, we propose a computational framework to quantify the extent of disorder within a coiled-coil in solution and to help design substitutions modulating such disorder. Our method relies on the analysis of conformational ensembles generated by relatively short all-atom Molecular Dynamics (MD) simulations. We apply it to the phosphoprotein multimerisation domains (PMD) of Measles virus (MeV) and Nipah virus (NiV), both forming tetrameric left-handed coiled-coils. We show that our method can help quantify the extent of disorder of the C-terminus region of MeV and NiV PMDs from MD simulations of a few tens of nanoseconds, and without requiring an extensive exploration of the conformational space. Moreover, this study provided a conceptual framework for the rational design of substitutions aimed at modulating the stability of the coiled-coils. By assessing the impact of four substitutions known to destabilize coiled-coils, we derive a set of rules to control MeV PMD structural stability and cohesiveness. We therefore design two contrasting substitutions, one increasing the stability of the tetramer and the other increasing its flexibility. CONCLUSIONS: Our method can be considered as a platform to reason about how to design substitutions aimed at regulating flexibility and stability.