A human antibody potently neutralizes RSV by targeting the conserved hydrophobic region of prefusion F.

Respiratory syncytial virus (RSV) continues to pose serious threats to pediatric populations due to the lack of a vaccine and effective antiviral drugs. RSV fusion (F) glycoprotein mediates viral-host membrane fusion and is a key target for neutralizing antibodies. We generated 23 full-human monoclonal antibodies (hmAbs) against prefusion F protein (pre-F) from a healthy adult with natural RSV infection by single B cell cloning technique. A highly potent RSV-neutralizing hmAb, named as 25-20, is selected, which targets a new site Ø-specific epitope. Site-directed mutagenesis and structural modelling analysis demonstrated that 25-20 mainly targets a highly conserved hydrophobic region located at the a4 helix and a1 helix of pre-F, indicating a site of vulnerability for drug and vaccine design. It is worth noting that 25-20 uses an unreported inferred germline (iGL) that binds very poorly to pre-F, thus high levels of somatic mutations are needed to gain high binding affinity with pre-F. Our observation helps to understand the evolution of RSV antibody during natural infection. Furthermore, by in silico prediction and experimental verification, we optimized 25-20 with KD values as low as picomolar range. Therefore, the optimized 25-20 represents an excellent candidate for passive protection against RSV infection.

Structural modeling, energetic analysis and molecular design of a π-stacking system at the complex interface of pediatric respiratory syncytial virus nucleocapsid with the C-terminal peptide of phosphoprotein.

Human respiratory syncytial virus (RSV) is a primary cause of lower respiratory tract infections and hospital visits during infancy and childhood. The RSV phosphoprotein (P) is a major polymerase cofactor that interacts with nucleoprotein (N) to promote the recognition of ribonucleoprotein complex (RNP) by viral RNA polymerase. The binding pocket of N protein is chemically diverse, in or around which a number of aromatic and charged amino acid residues are observed. Previously, a nonapeptide segment (P peptide, 233DNDLSLEDF241) representing the C-terminal tail of P protein was identified to mediate the N-P interaction with a moderate affinity, in which the Phe241 at the end of P's C-terminus plays a critical role in the binding of P peptide to N protein. Here, we found that the side-chain aromatic phenyl moiety of P Phe241 residue can form short- and long-range cation-π interactions with N Arg132 and Arg150 residues, respectively, as well as T-shaped and parallel-displaced π-π stackings with N Tyr135 and His151 residues, respectively, which co-define a geometrically satisfactory π-stacking system at the complex interface of N protein with P peptide, thus largely stabilizing the complex architecture. The stacking effect was further optimized by systematically mutating the P Phe241 residue to other natural and non-natural aromatic amino acids with diverse chemical substitutions at the phenyl moiety to examine their structural and energetic effects on π-stacking system and on protein-peptide binding. The electron-donating mutations at the phenyl moiety of P Phe241 residue can effectively enhance the π-stacking system and then promote peptide binding, whereas the bulky and positively charged mutations would considerably impair the peptide potency by introducing steric hindrance and electrostatic repulsion. The [Tyr]P, [Thp]P and [Fph]P mutants were determined to have an increased affinity relative to wild-type P peptide, which could be used as self-inhibitory peptides to competitively disrupt the native interaction between N and P proteins.

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein.

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 µM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection. We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

Biophysical studies of the interaction of hRSV Non-Structural 1 protein with natural flavonoids and their acetylated derivatives by spectroscopic techniques and computational simulations.

Human respiratory syncytial virus (hRSV) infections are one of the most causes of acute lower respiratory tract infections in children and elderly. The development of effective antiviral therapies or preventive vaccines against hRSV is not available yet. Thus, it is necessary to search for protein targets to combat this viral infection, as well as potential ways to block them. Non-Structural 1 (NS1) protein is an important factor for viral replication success since reduces the immune response by interacting with proteins in the type I interferon pathway. The influence of NS1 on the cell's immune response denotes the potential of its inhibition, being a possible target of treatment against hRSV infection. Here, it was studied the interaction of hRSV NS1 with natural flavonoids chrysin, morin, kaempferol, and myricetin and their mono-acetylated chrysin and penta-acetylated morin derivatives using spectroscopic techniques and computational simulations. The fluorescence data indicate that the binding affinities are on the order of 105 M-1, which are directly related to the partition coefficient of each flavonoid with Pearson's correlation coefficients of 0.76-0.80. The thermodynamic analysis suggests that hydrophobic interactions play a key role in the formation of the NS1/flavonoid complexes, with positive values of enthalpy and entropy changes. The computational approach proposes that flavonoids bind in a region of NS1 formed between the C-terminal α3-helix and the protein core, important for its biological function, and corroborate with experimental data revealing that hydrophobic contacts are important for the binding. Therefore, the present study provides relevant molecular details for the development of a possible new strategy to fight infections caused by hRSV.

Hesperetin targets the hydrophobic pocket of the nucleoprotein/phosphoprotein binding site of human respiratory syncytial virus.

The human Respiratory Syncytial Virus (hRSV) is one of the most common causes of acute respiratory diseases such as bronchiolitis and pneumonia in children worldwide. Among the viral proteins, the nucleoprotein (N) stands out for forming the nucleocapsid (NC) that functions as a template for replication and transcription by the viral polymerase complex. The NC/polymerase recognition is mediated by the phosphoprotein (P), which establishes an interaction of its C-terminal residues with a hydrophobic pocket in the N-terminal domain of N (N-NTD). The present study consists of biophysical characterization of N-NTD and investigation of flavonoids binding to this domain using experimental and computational approaches. Saturation transfer difference (STD)-NMR measurements showed that among the investigated flavonoids, only hesperetin (Hst) bound to N-NTD. The binding epitope mapping of Hst suggested that its fused aromatic ring is buried in the protein binding site. STD-NMR and fluorescence anisotropy experiments showed that Hst competes with P protein C-terminal dipeptides for the hRSV nucleoprotein/phosphoprotein (N/P) interaction site in N-NTD, indicating that Hst binds to the hydrophobic pocket in this domain. Computational simulations of molecular docking and dynamics corroborated with experimental results, presenting that Hst established a stable interaction with the N/P binding site. The outcomes presented herein shed light on literature reports that described a significant antireplicative activity of Hst against hRSV, revealing molecular details that can provide the development of a new strategy against this virus.

Helical ordering of envelope-associated proteins and glycoproteins in respiratory syncytial virus.

Human respiratory syncytial virus (RSV) causes severe respiratory illness in children and the elderly. Here, using cryogenic electron microscopy and tomography combined with computational image analysis and three-dimensional reconstruction, we show that there is extensive helical ordering of the envelope-associated proteins and glycoproteins of RSV filamentous virions. We calculated a 16 Å resolution sub-tomogram average of the matrix protein (M) layer that forms an endoskeleton below the viral envelope. These data define a helical lattice of M-dimers, showing how M is oriented relative to the viral envelope. Glycoproteins that stud the viral envelope were also found to be helically ordered, a property that was coordinated by the M-layer. Furthermore, envelope glycoproteins clustered in pairs, a feature that may have implications for the conformation of fusion (F) glycoprotein epitopes that are the principal target for vaccine and monoclonal antibody development. We also report the presence, in authentic virus infections, of N-RNA rings packaged within RSV virions. These data provide molecular insight into the organisation of the virion and the mechanism of its assembly.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

Functional Features of the Respiratory Syncytial Virus G Protein.

Respiratory syncytial virus (RSV) is a major cause of serious lower respiratory tract infections in children <5 years of age worldwide and repeated infections throughout life leading to serious disease in the elderly and persons with compromised immune, cardiac, and pulmonary systems. The disease burden has made it a high priority for vaccine and antiviral drug development but without success except for immune prophylaxis for certain young infants. Two RSV proteins are associated with protection, F and G, and F is most often pursued for vaccine and antiviral drug development. Several features of the G protein suggest it could also be an important to vaccine or antiviral drug target design. We review features of G that effect biology of infection, the host immune response, and disease associated with infection. Though it is not clear how to fit these together into an integrated picture, it is clear that G mediates cell surface binding and facilitates cellular infection, modulates host responses that affect both immunity and disease, and its CX3C aa motif contributes to many of these effects. These features of G and the ability to block the effects with antibody, suggest G has substantial potential in vaccine and antiviral drug design.

A Structural and Dynamic Analysis of the Partially Disordered Polymerase-Binding Domain in RSV Phosphoprotein.

The phosphoprotein P of Mononegavirales (MNV) is an essential co-factor of the viral RNA polymerase L. Its prime function is to recruit L to the ribonucleocapsid composed of the viral genome encapsidated by the nucleoprotein N. MNV phosphoproteins often contain a high degree of disorder. In Pneumoviridae phosphoproteins, the only domain with well-defined structure is a small oligomerization domain (POD). We previously characterized the differential disorder in respiratory syncytial virus (RSV) phosphoprotein by NMR. We showed that outside of RSV POD, the intrinsically disordered N-and C-terminal regions displayed a structural and dynamic diversity ranging from random coil to high helical propensity. Here we provide additional insight into the dynamic behavior of PCα, a domain that is C-terminal to POD and constitutes the RSV L-binding region together with POD. By using small phosphoprotein fragments centered on or adjacent to POD, we obtained a structural picture of the POD-PCα region in solution, at the single residue level by NMR and at lower resolution by complementary biophysical methods. We probed POD-PCα inter-domain contacts and showed that small molecules were able to modify the dynamics of PCα. These structural properties are fundamental to the peculiar binding mode of RSV phosphoprotein to L, where each of the four protomers binds to L in a different way.

Potent Human Single-Domain Antibodies Specific for a Novel Prefusion Epitope of Respiratory Syncytial Virus F Glycoprotein.

Respiratory syncytial virus (RSV) poses great health threats to humans. However, there are no licensed vaccines or therapeutic drugs to date. Only one humanized monoclonal antibody, palivizumab, is available on the market, but it is used prophylactically and is limited to infants with high risk. With advances in antibody engineering, it has been found that a single-domain antibody (sdAb) can be therapeutically administered by inhalation, which would be more efficient for respiratory diseases. Here, we identified two human sdAbs, m17 and m35, by phage display technology. They specifically bind to RSV fusion glycoprotein (F protein) in the prefusion state with subnanomolar affinity and potently neutralize both RSV subtypes A and B with 50% inhibitory concentration (IC50) values ranging from pM to nM. Interestingly, these sdAbs recognize a novel epitope, termed VI, that is unique to the prefusion state. This epitope is located at the C terminus of the F1 subunit, close to the viral membrane, and might be sterically restricted. We further find that m17 and m35 neutralize RSV by preventing the prefusion F conformational arrangement, thus inhibiting membrane fusion. These two sdAbs have the potential to be further developed as therapeutic candidates and may also provide novel insight for developing other antiviral reagents against RSV. IMPORTANCE Because respiratory syncytial virus (RSV) can cause serious respiratory disease in immunodeficient groups, including infants and seniors, the development of vaccines and therapeutic drugs, such as neutralizing antibodies, is urgently needed. Compared to the conventional full-length antibody, a single-domain antibody (sdAb) has been demonstrated to be efficient for respiratory diseases when administered by inhalation, thereby potentially introducing a kind of novel therapeutic agent in the market. Here, we discovered two potent neutralizing human sdAbs against RSV that recognized a novel prefusion epitope, termed VI, and prevented conformational arrangement during the fusion process. Our work provides not only therapeutic candidates but also novel targets for new drug and vaccine development.

Targeting the membrane fusion event of human respiratory syncytial virus with rationally designed α-helical hairpin traps.

AIMS: Rational design of protein scaffolds with specific biological functions/activities has attracted much attention over the past decades. In the present study, we systematically examine the trimer-of-hairpins (TOH) motif of human respiratory syncytial virus (RSV) F protein, which plays a central role in viral membrane fusion and is a coiled-coil six-helix bundle formed by the antiparallel intermolecular interaction between three N-terminal heptad-repeat (HRN) helices and three C-terminal heptad-repeat (HRC) helices. MAIN METHODS: A rational strategy that integrates dynamics simulation, thermodynamics calculation, fluorescence polarization and circular dichroism is proposed to design HRC-targeted α-helical hairpin traps based on the crystal template of HRN core. KEY FINDINGS: The designed hairpin traps possess a typical helix-turn-helix scaffold that can be stabilized by stapling a disulfide bridge across its helical arms, which are highly structured (helicity >60%) and can mimic the native spatial arrangement of HRN helices in TOH motif to trap the hotspot sites of HRC with effective affinity (Kd is up to 6.4 µM). SIGNIFICANCE: The designed α-helical hairpin traps can be used as lead entities for further developing TOH-disrupting agents to target RSV membrane fusion event and the proposed rational design strategy can be readily modified to apply for other type I viruses.

Structural Insights into the Respiratory Syncytial Virus RNA Synthesis Complexes.

RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (-) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5'-RNA capping and methylation, and 3' end polyadenylation. Genome replication includes (+) RNA antigenome and (-) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies.

Respiratory syncytial virus (RSV) infection can cause bronchiolitis, pneumonia, morbidity, and some mortality, primarily in infants and the elderly, for which no vaccine is available. The RSV attachment (G) protein contains a central conserved domain (CCD) with a CX3C motif implicated in the induction of protective antibodies, thus vaccine candidates containing the G protein are of interest. This study determined if mutations in the G protein CCD would mediate immunogenicity while inducing G protein CX3C-CX3CR1 blocking antibodies. BALB/c mice were vaccinated with structurally-guided, rationally designed G proteins with CCD mutations. The results show that these G protein immunogens induce a substantial anti-G protein antibody response, and using serum IgG from the vaccinated mice, these antibodies are capable of blocking the RSV G protein CX3C-CX3CR1 binding while not interfering with CX3CL1, fractalkine.

Structural Characterization and Modeling of a Respiratory Syncytial Virus Fusion Glycoprotein Nanoparticle Vaccine in Solution.

The respiratory syncytial virus (RSV) fusion (F) protein/polysorbate 80 (PS80) nanoparticle vaccine is the most clinically advanced vaccine for maternal immunization and protection of newborns against RSV infection. It is composed of a near-full-length RSV F glycoprotein, with an intact membrane domain, formulated into a stable nanoparticle with PS80 detergent. To understand the structural basis for the efficacy of the vaccine, a comprehensive study of its structure and hydrodynamic properties in solution was performed. Small-angle neutron scattering experiments indicate that the nanoparticle contains an average of 350 PS80 molecules, which form a cylindrical micellar core structure and five RSV F trimers that are arranged around the long axis of the PS80 core. All-atom models of full-length RSV F trimers were built from crystal structures of the soluble ectodomain and arranged around the long axis of the PS80 core, allowing for the generation of an ensemble of conformations that agree with small-angle neutron and X-ray scattering data as well as transmission electron microscopy (TEM) images. Furthermore, the hydrodynamic size of the RSV F nanoparticle was found to be modulated by the molar ratio of PS80 to protein, suggesting a mechanism for nanoparticle assembly involving addition of RSV F trimers to and growth along the long axis of the PS80 core. This study provides structural details of antigen presentation and conformation in the RSV F nanoparticle vaccine, helping to explain the induction of broad immunity and observed clinical efficacy. Small-angle scattering methods provide a general strategy to visualize surface glycoproteins from other pathogens and to structurally characterize nanoparticle vaccines.

A respiratory syncytial virus (RSV) F protein nanoparticle vaccine focuses antibody responses to a conserved neutralization domain.

A stabilized form of the respiratory syncytial virus (RSV) fusion (F) protein has been explored as a vaccine to prevent viral infection because it presents several potent neutralizing epitopes. Here, we used a structure-based rational design to optimize antigen presentation and focus antibody (Ab) responses to key epitopes on the pre-fusion (pre-F) protein. This protein was fused to ferritin nanoparticles (pre-F-NP) and modified with glycans to mask nonneutralizing or poorly neutralizing epitopes to further focus the Ab response. The multimeric pre-F-NP elicited durable pre-F-specific Abs in nonhuman primates (NHPs) after >150 days and elicited potent neutralizing Ab (NAb) responses in mice and NHPs in vivo, as well as in human cells evaluated in the in vitro MIMIC system. This optimized pre-F-NP stimulated a more potent Ab response than a representative pre-F trimer, DS-Cav1. Collectively, this pre-F vaccine increased the generation of NAbs targeting the desired pre-F conformation, an attribute that facilitates the development of an effective RSV vaccine.

In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus.

The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N-RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.

Alternative conformations of a major antigenic site on RSV F.

The respiratory syncytial virus (RSV) fusion (F) glycoprotein is a major target of neutralizing antibodies arising from natural infection, and antibodies that specifically bind to the prefusion conformation of RSV F generally demonstrate the greatest neutralization potency. Prefusion-stabilized RSV F variants have been engineered as vaccine antigens, but crystal structures of these variants have revealed conformational differences in a key antigenic site located at the apex of the trimer, referred to as antigenic site Ø. Currently, it is unclear if flexibility in this region is an inherent property of prefusion RSV F or if it is related to inadequate stabilization of site Ø in the engineered variants. Therefore, we set out to investigate the conformational flexibility of antigenic site Ø, as well as the ability of the human immune system to recognize alternative conformations of this site, by determining crystal structures of prefusion RSV F bound to neutralizing human-derived antibodies AM22 and RSD5. Both antibodies bound with high affinity and were specific for the prefusion conformation of RSV F. Crystal structures of the complexes revealed that the antibodies recognized distinct conformations of antigenic site Ø, each diverging at a conserved proline residue located in the middle of an α-helix. These data suggest that antigenic site Ø exists as an ensemble of conformations, with individual antibodies recognizing discrete states. Collectively, these results have implications for the refolding of pneumovirus and paramyxovirus fusion proteins and should inform development of prefusion-stabilized RSV F vaccine candidates.

Discovery of Ziresovir as a Potent, Selective, and Orally Bioavailable Respiratory Syncytial Virus Fusion Protein Inhibitor.

Ziresovir (RO-0529, AK0529) is reported here for the first time as a promising respiratory syncytial virus (RSV) fusion (F) protein inhibitor that currently is in phase 2 clinical trials. This article describes the process of RO-0529 as a potent, selective, and orally bioavailable RSV F protein inhibitor and highlights the in vitro and in vivo anti-RSV activities and pharmacokinetics in animal species. RO-0529 demonstrates single-digit nM EC50 potency against laboratory strains, as well as clinical isolates of RSV in cellular assays, and more than one log viral load reduction in BALB/c mouse model of RSV viral infection. RO-0529 was proven to be a specific RSV F protein inhibitor by identification of drug resistant mutations of D486N, D489V, and D489Y in RSV F protein and the inhibition of RSV F protein-induced cell-cell fusion in cellular assays.

Conformational Isomerization Involving Conserved Proline Residues Modulates Oligomerization of the NS1 Interferon Response Inhibitor from the Syncytial Respiratory Virus.

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main ß-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes.