Viral Fusion Proteins Research Articles

Examination of respiratory syncytial virus fusion protein proteolytic processing and roles of the P27 domain.

The respiratory syncytial virus (RSV) fusion protein (F) facilitates virus-cell membrane fusion, which is critical for viral entry, and cell-cell fusion. In contrast to many type I fusion proteins, RSV F must be proteolytically cleaved at two distinct sites to be fusogenic. Cleavage at both sites results in the release of a 27 amino-acid fragment, termed Pep27. We examined proteolytic processing and the role of Pep27 for RSV F from both RSV A2 and RSV B9320 laboratory-adapted strains, allowing important comparisons between A and B clade F proteins. F from both clades was cleaved at both sites, and pulse-chase analysis indicated that cleavage at both sites occurs early after synthesis, most likely within the secretory pathway. Mutation of either site to alter the furin recognition motif blocked cell-cell fusion activity. To assess the role of Pep27 in F processing and expression, we deleted the Pep27 fragment, but preserved the cleavage sites. Deletion of Pep27 reduced F surface expression and cell-cell fusion. Two conserved N-linked glycosylation sites within Pep 27 are present in both the RSV A2 and RSV B9320 F. Randomization of the Pep27 sequence, while conserving the two N-liked glycosylation sites, did not significantly change surface expression, and only modestly reduced cell-cell fusion. However, the disruption of either Pep27 glycosylation site reduced cell-cell fusion. This work clarifies the timing of RSV F proteolytic cleavage and offers insight into the crucial role the N-linked glycosylation sites within Pep27 play in the biological function of F.

Developing a chimeric multiepitope vaccine against Nipah virus (NiV) through immunoinformatics, molecular docking and dynamic simulation approaches

Nipah virus (NiV) is a highly lethal zoonotic pathogen that poses a significant threat to human and animal health. Unfortunately, no effective treatments have been developed for this deadly zoonotic disease. Therefore, we designed a chimeric multiepitope vaccine targeting the Nipah virus (NiV) glycoprotein and fusion protein through immunoinformatic approaches. Therefore, the vaccine was developed by combining promising and potential antigenic MHC-I, MHC-II, and B-cell epitopes obtained from the selected proteins. When combined, the MHC-I and MHC-II epitopes offered 100% global population coverage. The physicochemical characterization also exhibited favorable properties, including solubility and potential functional stability of the vaccine within the body (GRAVY score of -0.308). Structural analyses unveiled a well-stabilized secondary and tertiary structure with a Ramachandran score of 84.4% and a Z score of -5.02. Findings from docking experiments with TLR-2 (-1089.3 KJ/mol) and TLR-4 (-1016.7 KJ/mol) showed a strong affinity of the vaccine towards the receptor. Molecular dynamics simulations revealed unique conformational dynamics among the "vaccine-apo," "vaccine-TLR-2," and "vaccine-TLR-4" complexes. Consequently, the complexes exhibited significant compactness, flexibility, and exposure to solvents. The results of the codon optimization were remarkable, as the vaccine showed a significant amount of expression in the E. coli vector (GC content of 45.36 % and a CAI score of 1.0). The results of immune simulations, however, showed evidence of both adaptive and innate immune responses induced by the vaccine. Therefore, we highly recommend further research on this chimeric multiepitope vaccine to establish its efficacy and safety against the Nipah virus (NiV).

The Cumulative Variations of Respiratory Syncytial Virus Fusion Protein (F) in Ten Consecutive Years in China.

Variations in the fusion (F) protein of respiratory syncytial virus (RSV) with main antigenic sites I-V and Ø may affect the development of RSV vaccines and therapies. In the study, 30 respiratory specimens positive for RSV were randomly selected from children with acute lower respiratory infections (ALRI) in Beijing every year from 2012 to 2021 for F gene sequencing. Then, 300 F gene sequences and 508 uploaded to GenBank from China were subjected to phylogenetic analysis. The results indicated the nucleotide identities were 95.4-100% among 446 sequences of RSV A, and 96.3-100% among 362 of RSV B. The most common variant loci were N80K (100.00%) and R213S (97.76%) for site Ø, and V384I/T (98.43%) for site I among sequences of RSV A, and M152I (100.00%), I185V (100.00%), and L172Q/H (94.48%) for site V, and R202Q (99.45%) for site Ø among sequences of RSV B. N276S appears in 95.29% sequences of RSV A, while S276N and N262 I/S appear in 1.38% and 0.55% sequences of RSV B, respectively. No variation was found in all sequences at the binding sites of 14N4 and motavizumab. There were cumulative variations of the RSV F gene, especially at some binding sites of antigenic sites.

Structures of the Foamy virus fusion protein reveal an unexpected link with the F protein of paramyxo- and pneumoviruses.

Foamy viruses (FVs) constitute a subfamily of retroviruses. Their envelope (Env) glycoprotein drives the merger of viral and cellular membranes during entry into cells. The only available structures of retroviral Envs are those from human and simian immunodeficiency viruses from the subfamily of orthoretroviruses, which are only distantly related to the FVs. We report the cryo-electron microscopy structures of the FV Env ectodomain in the pre- and post-fusion states, which unexpectedly demonstrate structural similarity with the fusion protein (F) of paramyxo- and pneumoviruses, implying an evolutionary link between the viral fusogens. We describe the structural features that are unique to the FV Env and propose a mechanistic model for its conformational change, highlighting how the interplay of its structural elements could drive membrane fusion and viral entry. The structural knowledge on the FV Env now provides a framework for functional investigations, which can benefit the design of FV Env variants with improved features for use as gene therapy vectors.

Molecular detection of lumpy skin disease virus in naturally infected cattle and buffaloes: unveiling the role of tick vectors in disease spread.

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals.

Engineering a cleaved, prefusion-stabilized influenza B virus hemagglutinin by identification and locking of all six pH switches.

Vaccine components based on viral fusion proteins require high stability of the native prefusion conformation for optimal potency and manufacturability. In the case of influenza B virus hemagglutinin (HA), the stem's conformation relies on efficient cleavage. In this study, we identified six pH-sensitive regions distributed across the entire ectodomain where protonated histidines assume either a repulsive or an attractive role. Substitutions in these areas enhanced the protein's expression, quality, and stability in its prefusion trimeric state. Importantly, this stabilization enabled the production of a cleavable HA0, which is further processed into HA1 and HA2 by furin during exocytic pathway passage, thereby facilitating correct folding, increased stability, and screening for additional stabilizing substitutions in the core of the metastable fusion domain. Cryo-EM analysis at neutral and low pH revealed a previously unnoticed pH switch involving the C-terminal residues of the natively cleaved HA1. This switch keeps the fusion peptide in a clamped state at neutral pH, averting premature conformational shift. Our findings shed light on new strategies for possible improvements of recombinant or genetic-based influenza B vaccines.

Strategic Design of Tryptophan-Aspartic Acid-Containing Peptide Inhibitors Using Coronin 1 as a Template: Inhibition of Fusion by Enhancing Acyl Chain Order.

Enveloped viruses enter the host cell by fusing at the cell membrane or entering the cell via endocytosis and fusing at the endosome. Conventional inhibitors target the viral fusion protein to inactivate it for inducing fusion. These target-specific vis-à-vis virus-specific inhibitors fail to display their inhibitory efficacy against emerging and remerging viral infections. This necessitates the need to develop broad-spectrum entry inhibitors that are effective irrespective of the virus. Using a broad range of targeting techniques, the fusion inhibitors can modify the physical characteristics of the viral membrane, making it less prone to fusion. We have previously shown that two tryptophan-aspartic acid (WD)-containing hydrophobic peptides, TG-23 and GG-21, from coronin 1, a phagosomal protein, inhibit membrane fusion by modulating membrane organization and dynamics. In the present work, we designed two WD-containing hydrophilic peptides, QG-22 and AG-22, using coronin 1 as a template and evaluated their fusion inhibitory efficacies in the absence and presence of membrane cholesterol. Our results demonstrate that QG-22 and AG-22 inhibit membrane fusion irrespective of the concentration of membrane cholesterol. Our measurements of depth-dependent membrane organization and dynamics reveal that they impede fusion by enhancing the acyl chain order. Overall, our results validate the hypothesis of designing fusion inhibitors by modulating the membrane's physical properties. In addition, it demonstrates that chain hydrophobicity might not be a critical determinant for the development of peptide-based fusion inhibitors.

Structural transition of GP64 triggered by a pH-sensitive multi-histidine switch

The fusion of viruses with cellular membranes is a critical step in the life cycle of enveloped viruses. This process is facilitated by viral fusion proteins, many of which are conformationally pH-sensitive. The specifics of how changes in pH initiate this fusion have remained largely elusive. This study presents the cryo-electron microscopy (cryo-EM) structures of a prototype class III fusion protein, GP64, in its prefusion and early intermediate states, revealing the structural intermediates accompanying the membrane fusion process. The structures identify the involvement of a pH-sensitive switch, comprising H23, H245, and H304, in sensing the low pH that triggers the initial step of membrane fusion. The pH sensing role of this switch is corroborated by assays of cell-cell syncytium formation and dual dye-labeling. The findings demonstrate that coordination between multiple histidine residues acts as a pH sensor and activator. The involvement of a multi-histidine switch in viral fusion is applicable to fusogens of human-infecting thogotoviruses and other viruses, which could lead to strategies for developing anti-viral therapies and vaccines.

A new mechanism of respiratory syncytial virus entry inhibition by small-molecule to overcome K394R-associated resistance.

Respiratory syncytial virus (RSV) infection is a major public health concern, and many small-molecule candidates targeting the viral fusion (F) protein are associated with a considerable risk of inducing drug-resistant mutations. This study investigated virological features of the K394R variant, a mutant strain conferring resistance to multiple RSV fusion inhibitors. Our results demonstrated that the K394R variant is highly fusogenic in cell cultures and more pathogenic than the parental strain in mice. The small-molecule inhibitor CL-A3-7 substantially reduced in vitro and in vivo infections of both wild-type RSV and the K394R variant by blocking the interaction of viral F protein with its cellular receptor, showing a new mechanism of action for small-molecules to inhibit RSV infection and overcome K394R-associated resistance.

From antibiotic to antiviral: computational screening reveals a multi-targeting antibiotic from Streptomyces spp. against Nipah virus fusion proteins.

Nipah Virus is a re-emerging zoonotic paramyxovirus that poses a significant threat to both swine industry and human health. The pursuit of potential antiviral agents with both preventive and therapeutic properties holds promise for targeting such viruses. To expedite this search, leveraging computational biology is essential. Streptomyces is renowned for its capacity to produce large and diverse metabolites with promising bioactivities. In the current study, we conducted a comprehensive structure-based virtual screening of 6524 Streptomyces spp. metabolites sourced from the StreptomeDB database to evaluate their potential inhibitory effects on three Nipah virus fusion (NiVF) protein conformations: NiVF pre-fusion 1-mer (NiVF-1mer), pre-fusion 3-mer (NiVF-3mer), and NiVF post-fusion (NiVF-PoF). Prior to virtual screening, the drug-likeness of Streptomyces spp. compounds was profiled using ADMET properties. From the 913 ADMET-filtered compounds, the subsequent targeted and confirmatory blind docking analysis revealed that S896 or virginiamycin M1, a known macrolide antibiotic, showed a maximum binding affinity with the NiVF proteins, suggesting a multi-targeting inhibitory property. In addition, the 200-ns molecular dynamics simulation and MM/PBSA analyses revealed stable and strong binding affinity between the NiVF-S896 complexes, indicating favorable interactions between S896 and the target proteins. These findings suggest the potential of virginiamycin M1, an antibiotic, as a promising multi-targeting antiviral drug. However, in vitro and in vivo experimental validations are necessary to assess their safety and efficacy.

Exploiting the Affimer platform against influenza A virus.

Influenza A virus is one of the few viruses that can cause devastating pandemics. Due to the high mutation rates of this virus, annual vaccination is required, and antivirals are short-lived. Monoclonal antibodies present a promising approach to tackle influenza virus infections but are associated with some limitations. To improve on this strategy, we explored the Affimer platform, which are antibody-like proteins made in bacteria. By performing phage-display against a monomeric version of influenza virus fusion protein, an established viral target, we were able to isolate Affimers that inhibit influenza virus infection in vitro. We characterized the mechanism of inhibition of the Affimers by using assays targeting different stages of the viral replication cycle. We additionally characterized HA-Affimer complex structure, using a novel approach to prepare samples for cryo-electron microscopy. Overall, these results show that Affimers are a promising tool against influenza virus infection.

Antibody levels against respiratory syncytial virus fusion protein conformations and lack of association with life-threatening infection in previously healthy infants

BackgroundHumoral immune response against the pre-fusion (pre-F) conformation of respiratory syncytial virus (RSV) F protein has been proposed to play a protective role against infection. An RSV pre-F maternal vaccine has been recently approved in several countries to protect young infants against RSV. We aimed to assess serum IgG titers against the pre-F and post-F conformations of RSV F protein and their association with life-threatening RSV disease (LTD) in previously healthy infants. MethodsA prospective cohort study including hospitalized infants <12 months with a first RSV infection was conducted during 2017–2019. Patients with LTD required intensive care and mechanical respiratory assistance. RSV pre-F exclusive and post-F antibody responses were determined by post-F competition and non-competition immunoassays, respectively, and neutralizing activity was measured by plaque reduction neutralization test. ResultsFifty-eight patients were included; the median age was 3.5 months and 41 % were females. Fifteen patients developed LTD. RSV F-specific antibody titers positively correlated with neutralizing antibody titers in acute and convalescent phases but, importantly, they did not associate with LTD. Acute RSV pre-F exclusive and post-F IgG titers negatively correlated with patient age (P = 0.0007 and P < 0.0001), while a positive correlation was observed between the fold changes in RSV F-specific antibody titers between convalescent and acute phase and patient age (P = 0.0014 and P < 0.0001). Infants ≤2 months exhibited significantly lower fold-changes in RSV F-specific and neutralizing antibody titers between convalescence and acute phase than older infants. Additionally, acute RSV antibody titers showed no correlation with nasal RSV load and, furthermore, nasal viral load was not associated with the development of LTD. ConclusionsThis study highlights that protection against life-threatening RSV disease is not necessarily antibody-dependent. Further characterization of the immune response against RSV and its role in protection against severe disease is important for the development of the safest possible preventive strategies.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

Structure and Interactions of HIV-1 gp41 CHR-NHR Reverse Hairpin Constructs Reveal Molecular Determinants of Antiviral Activity

Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.

Emodin inhibits respiratory syncytial virus entry by interactions with fusion protein.

Respiratory syncytial virus (RSV) fusion (F) protein is essential for facilitating virus entry into host cells, providing a hopeful path for combating viral diseases. However, F protein inhibitors can rapidly select for viral resistance. Thus, discovering new inhibitors of F-protein is necessary to enrich the RSV drug development pipeline. In this study, we screen 25 bioactive compounds from Chinese herbal medicines that exhibit a strong binding to the RSV-F protein using surface plasmon resonance. After screening, we found emodin could strongly bind to RSV-F protein, and could effectively curb RSV infection. Further investigations certificated that emodin specifically disrupts the attachment and internalization phases of RSV infection by targeting the RSV-F protein. In vivo studies with mice infected with RSV demonstrated that emodin effectively reduces lung pathology. This therapeutic effect is attributed to emodin's capacity to diminish pro-inflammatory cytokine production and reduce viral load in the lungs. In conclusion, our findings provide initial insights into the mechanism by which emodin counters RSV infection via engagement with the RSV-F protein, establishing it as a viable contender for the development of novel therapeutic agents aimed at RSV.

Monoclonal antibodies targeting sites in respiratory syncytial virus attachment G protein provide protection against RSV-A and RSV-B in mice

Currently, only Palivizumab and Nirsevimab that target the respiratory syncytical virus (RSV) fusion protein are licensed for pre-treatment of infants. Glycoprotein-targeting antibodies may also provide protection against RSV. In this study, we generate monoclonal antibodies from mice immunized with G proteins from RSV-A2 and RSV-B1 strains. These monoclonal antibodies recognize six unique antigenic classes (G0-G5). None of the anti-G monoclonal antibodies neutralize RSV-A2 or RSV-B1 in vitro. In mice challenged with either RSV-A2 line 19 F or RSV-B1, one day after treatment with anti-G monoclonal antibodies, all monoclonal antibodies reduce lung pathology and significantly reduce lung infectious viral titers by more than 2 logs on day 5 post-RSV challenge. RSV dissemination in the lungs was variable and correlated with lung pathology. We demonstrate new cross-protective anti-G monoclonal antibodies targeting multiple sites including conformation-dependent class G0 MAb 77D2, CCD-specific class G1 MAb 40D8, and carboxy terminus of CCD class G5 MAb 7H11, to support development of G-targeting monoclonal antibodies against RSV.

The rotavirus VP5*/VP8* conformational transition permeabilizes membranes to Ca2.

Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.

Engineered dityrosine-bonding of the RSV prefusion F protein imparts stability and potency advantages

Viral fusion proteins facilitate cellular infection by fusing viral and cellular membranes, which involves dramatic transitions from their pre- to postfusion conformations. These proteins are among the most protective viral immunogens, but they are metastable which often makes them intractable as subunit vaccine targets. Adapting a natural enzymatic reaction, we harness the structural rigidity that targeted dityrosine crosslinks impart to covalently stabilize fusion proteins in their native conformations. We show that the prefusion conformation of respiratory syncytial virus fusion protein can be stabilized with two engineered dityrosine crosslinks (DT-preF), markedly improving its stability and shelf-life. Furthermore, it has 11X greater potency as compared with the DS-Cav1 stabilized prefusion F protein in immunogenicity studies and overcomes immunosenescence in mice with simply a high-dose formulation on alum.

Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Drug repurposing screen identifies lonafarnib as respiratory syncytial virus fusion protein inhibitor.

Respiratory syncytial virus (RSV) is a common cause of acute lower respiratory tract infection in infants, older adults and the immunocompromised. Effective directly acting antivirals are not yet available for clinical use. To address this, we screen the ReFRAME drug-repurposing library consisting of 12,000 small molecules against RSV. We identify 21 primary candidates including RSV F and N protein inhibitors, five HSP90 and four IMPDH inhibitors. We select lonafarnib, a licensed farnesyltransferase inhibitor, and phase III candidate for hepatitis delta virus (HDV) therapy, for further follow-up. Dose-response analyses and plaque assays confirm the antiviral activity (IC50: 10-118 nM). Passaging of RSV with lonafarnib selects for phenotypic resistance and fixation of mutations in the RSV fusion protein (T335I and T400A). Lentiviral pseudotypes programmed with variant RSV fusion proteins confirm that lonafarnib inhibits RSV cell entry and that these mutations confer lonafarnib resistance. Surface plasmon resonance reveals RSV fusion protein binding of lonafarnib and co-crystallography identifies the lonafarnib binding site within RSV F. Oral administration of lonafarnib dose-dependently reduces RSV virus load in a murine infection model using female mice. Collectively, this work provides an overview of RSV drug repurposing candidates and establishes lonafarnib as a bona fide fusion protein inhibitor.