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.

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.

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.

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.

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.

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.

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.

Protective antigenic sites in respiratory syncytial virus G attachment protein outside the central conserved and cysteine noose domains.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants. Previously, we elucidated the antibody repertoire following primary RSV infection in infants. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes from RSV bound 100-fold more phages within attachment protein (G) following primary RSV infection. The G-reactive epitopes spanned the N- and C-termini of G ectodomain, in addition to the central conserved domain (CCD). In the current study, we examined the contribution of antigenic regions of G outside of the CCD to RSV-specific immunity. We evaluated the immunogenicity, neutralization and protective efficacy of all RSV-G antigenic sites identified following primary RSV infection using recombinant E. coli expressed G ectodomain (REG), CCD-deleted G ectodomain (REG ΔCCD), N- and C-terminal G subdomains, and antigenic site peptides. The REG ΔCCD, N- and C-terminal subdomains and peptides generated antibody titers in rabbits and mice that bound fully glycosylated Recombinant Mammalian expressed G ectodomain (RMG) and intact RSV virion particles but minimal in vitro neutralization titers compared with the intact G ectodomain. Vaccinated mice were challenged intranasally with RSV-A2 Line 19F. Viral replication in nasal cavity and lungs was significantly reduced in vaccinated animals compared to unimmunized controls. Control of viral loads post-RSV challenge correlated with serum antibody binding to the virus particles. In addition, very low Th2/Th1 cytokine ratios were found in the lungs of REG ΔCCD vaccinated mice after challenge. These data demonstrate the presence of multiple protective sites in RSV G protein outside of the CCD that could contribute to the development of a bacterially produced unglycosylated G protein as safe and protective vaccine against RSV disease.

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.

Evaluation of the role of respiratory syncytial virus surface glycoproteins F and G on viral stability and replication: implications for future vaccine design.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

A conformational switch balances viral RNA accessibility and protection in a nucleocapsid ring model.

Virus from the Mononegavirales order share common features ranging from virion structure arrangement to mechanisms of replication and transcription. One of them is the way the nucleoprotein (N) wraps and protects the RNA genome from degradation by forming a highly ordered helical nucleocapsid. However, crystal structures from numerous Mononegavirales reveal that binding to the nucleoprotein results in occluded nucleotides that hinder base pairing necessary for transcription and replication. This hints at the existence of alternative conformations of the N protein that would impact on the protein-RNA interface, allowing for transient exposure of the nucleotides without complete RNA release. Moreover, the regulation between the alternative conformations should be finely tuned. Recombinant expression of N from the respiratory syncytial virus form regular N/RNA common among all Mononegavirales, and these constitute an ideal minimal unit for investigating the mechanisms through which these structures protect RNA so efficiently while allowing for partial accessibility during transcription and replication. Neither pH nor high ionic strength could dissociate the RNA but led to irreversible aggregation of the nucleoprotein. Low concentrations of guanidine chloride dissociated the RNA moiety but leading to irreversible aggregation of the protein moiety. On the other hand, high concentrations of urea and long incubation periods were required to remove bound RNA. Both denaturants eventually led to unfolding but converged in the formation of an RNA-free ß-enriched intermediate species that remained decameric even at high denaturant concentrations. Although the N-RNA rings interact with the phosphoprotein P, the scaffold of the RNA polymerase complex, this interaction did not lead to RNA dissociation from the rings in vitro. Thus, we have uncovered complex equilibria involving changes in secondary structure of N and RNA loosening, processes that must take place in the context of RNA transcription and replication, whose detailed mechanisms and cellular and viral participants need to be established.

Structure and Function of the Human Respiratory Syncytial Virus M2-1 Protein.

Human respiratory syncytial virus (HRSV) is a non-segmented negative stranded RNA virus and is recognized as the most important viral agent of lower respiratory tract infection worldwide, responsible for up to 199,000 deaths each year. The only FDA-approved regime to prevent HRSV-mediated disease is pre-exposure administration of a humanized HRSV-specific monoclonal antibody, which although being effective, is not in widespread usage due to its cost. No HRSV vaccine exists and so there remains a strong need for alternative and complementary anti-HRSV therapies. The HRSV M2-1 protein is a transcription factor and represents an attractive target for the development of antiviral compounds, based on its essential role in the viral replication cycle. To this end, a detailed analysis of M2-1 structure and functions will aid in identifying rational targets for structure-based antiviral drug design that can be developed in future translational research. Here we present an overview of the current understanding of the structure and function of HRSV M2-1, drawing on additional information derived from its structural homologues from other related viruses.

A Randomized, Controlled, Observer-Blinded Phase 1 Study of the Safety and Immunogenicity of a Respiratory Syncytial Virus Vaccine With or Without Alum Adjuvant.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of childhood bronchiolitis and pneumonia, particularly in early infancy. Immunization of pregnant women could boost preexisting immune responses, providing passive protection to newborns through placental transfer of anti-RSV antibody. METHODS: In this first-in-humans clinical trial of a purified recombinant RSV protein F vaccine engineered to preferentially maintain prefusion conformation (RSV-PreF), 128 healthy men 18-44 years old were randomized to one dose of a RSV-PreF vaccine containing 10, 30, or 60 µg of RSV-PreF antigen, with or without alum adjuvant, or control, and followed for one year for safety and immunogenicity outcomes. RESULTS: Injection site pain was the most common adverse event, reported by up to 81.3% of participants. The highest RSV neutralizing antibody responses were in the 30 µg RSV-PreF/alum, 60 µg RSV-PreF/alum, and 60 µg RSV-PreF/nonadjuvant groups. Responses were evident on day 7, and 30 days after vaccination these participants had RSV-A neutralizing antibody titers of ≥1:512, and >70% had titers of 1:1024, with titers increasing by 3.2-4.9 fold. Responses remained high on day 60 but waned on days 180 and 360. CONCLUSIONS: The RSV-PreF vaccine elicited rapid RSV neutralizing antibody responses in healthy young men, with an acceptable adverse event profile.

A Short Double-Stapled Peptide Inhibits Respiratory Syncytial Virus Entry and Spreading.

Synthetic peptides derived from the heptad repeat (HR) of fusion (F) proteins can be used as dominant negative inhibitors to inhibit the fusion mechanism of class I viral F proteins. Here, we have performed a stapled-peptide scan across the HR2 domain of the respiratory syncytial virus (RSV) F protein with the aim to identify a minimal domain capable of disrupting the formation of the postfusion six-helix bundle required for viral cell entry. Constraining the peptides with a single staple was not sufficient to inhibit RSV infection. However, the insertion of double staples led to the identification of novel short stapled peptides that display nanomolar potency in HEp-2 cells and are exceptionally robust to proteolytic degradation. By replacing each amino acid of the peptides by an alanine, we found that the substitution of residues 506 to 509, located in a patch of polar contacts between HR2 and HR1, severely affected inhibition. Finally, we show that intranasal delivery of the most potent peptide to BALB/c mice significantly decreased RSV infection in upper and lower respiratory tracts. The discovery of this minimal HR2 sequence as a means for inhibition of RSV infection provides the basis for further medicinal chemistry efforts toward developing RSV fusion antivirals.

Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

UNLABELLED: Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. IMPORTANCE: RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

The viral transcription group determines the HLA class I cellular immune response against human respiratory syncytial virus.

The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.

Intrinsic Disorder to Order Transitions in the Scaffold Phosphoprotein P from the Respiratory Syncytial Virus RNA Polymerase Complex.

Intrinsic disorder is at the center of biochemical regulation and is particularly overrepresented among the often multifunctional viral proteins. Replication and transcription of the respiratory syncytial virus (RSV) relies on a RNA polymerase complex with a phosphoprotein cofactor P as the structural scaffold, which consists of a four-helix bundle tetramerization domain flanked by two domains predicted to be intrinsically disordered. Because intrinsic disorder cannot be reduced to a defined atomic structure, we tackled the experimental dissection of the disorder-order transitions of P by a domain fragmentation approach. P remains as a tetramer above 70 °C but shows a pronounced reversible secondary structure transition between 10 and 60 °C. While the N-terminal module behaves as a random coil-like IDP in a manner independent of tetramerization, the isolated C-terminal module displays a cooperative and reversible metastable transition. When linked to the tetramerization domain, the C-terminal module becomes markedly more structured and stable, with strong ANS binding. Therefore, the tertiary structure in the C-terminal module is not compact, conferring "late" molten globule-like IDP properties, stabilized by interactions favored by tetramerization. The presence of a folded structure highly sensitive to temperature, reversibly and almost instantly formed and broken, suggests a temperature sensing activity. The marginal stability allows for exposure of protein binding sites, offering a thermodynamic and kinetic fine-tuning in order-disorder transitions, essential for the assembly and function of the RSV RNA polymerase complex.

A Druggable Pocket at the Nucleocapsid/Phosphoprotein Interaction Site of Human Respiratory Syncytial Virus.

UNLABELLED: Presently, respiratory syncytial virus (RSV), the main cause of severe respiratory infections in infants, cannot be treated efficiently with antivirals. However, its RNA-dependent polymerase complex offers potential targets for RSV-specific drugs. This includes the recognition of its template, the ribonucleoprotein complex (RNP), consisting of genomic RNA encapsidated by the RSV nucleoprotein, N. This recognition proceeds via interaction between the phosphoprotein P, which is the main polymerase cofactor, and N. The determinant role of the C terminus of P, and more particularly of the last residue, F241, in RNP binding and viral RNA synthesis has been assessed previously. Here, we provide detailed structural insight into this crucial interaction for RSV polymerase activity. We solved the crystallographic structures of complexes between the N-terminal domain of N (N-NTD) and C-terminal peptides of P and characterized binding by biophysical approaches. Our results provide a rationale for the pivotal role of F241, which inserts into a well-defined N-NTD pocket. This primary binding site is completed by transient contacts with upstream P residues outside the pocket. Based on the structural information of the N-NTD:P complex, we identified inhibitors of this interaction, selected by in silico screening of small compounds, that efficiently bind to N and compete with P in vitro. One of the compounds displayed inhibitory activity on RSV replication, thereby strengthening the relevance of N-NTD for structure-based design of RSV-specific antivirals. IMPORTANCE: Respiratory syncytial virus (RSV) is a widespread pathogen that is a leading cause of acute lower respiratory infections in infants worldwide. RSV cannot be treated efficiently with antivirals, and no vaccine is presently available, with the development of pediatric vaccines being particularly challenging. Therefore, there is a need for new therapeutic strategies that specifically target RSV. The interaction between the RSV phosphoprotein P and the ribonucleoprotein complex is critical for viral replication. In this study, we identified the main structural determinants of this interaction, and we used them to screen potential inhibitors in silico. We found a family of molecules that were efficient competitors of P in vitro and showed inhibitory activity on RSV replication in cellular assays. These compounds provide a basis for a pharmacophore model that must be improved but that holds promises for the design of new RSV-specific antivirals.

Conservation of G-Protein Epitopes in Respiratory Syncytial Virus (Group A) Despite Broad Genetic Diversity: Is Antibody Selection Involved in Virus Evolution?

UNLABELLED: Worldwide G-glycoprotein phylogeny of human respiratory syncytial virus (hRSV) group A sequences revealed diversification in major clades and genotypes over more than 50 years of recorded history. Multiple genotypes cocirculated during prolonged periods of time, but recent dominance of the GA2 genotype was noticed in several studies, and it is highlighted here with sequences from viruses circulating recently in Spain and Panama. Reactivity of group A viruses with monoclonal antibodies (MAbs) that recognize strain-variable epitopes of the G glycoprotein failed to correlate genotype diversification with antibody reactivity. Additionally, no clear correlation was found between changes in strain-variable epitopes and predicted sites of positive selection, despite both traits being associated with the C-terminal third of the G glycoprotein. Hence, our data do not lend support to the proposed antibody-driven selection of variants as a major determinant of hRSV evolution. Other alternative mechanisms are considered to account for the high degree of hRSV G-protein variability. IMPORTANCE: An unusual characteristic of the G glycoprotein of human respiratory syncytial virus (hRSV) is the accumulation of nonsynonymous (N) changes at higher rates than synonymous (S) changes, reaching dN/dS values at certain sites predictive of positive selection. Since these sites cluster preferentially in the C-terminal third of the G protein, like certain epitopes recognized by murine antibodies, it was proposed that immune (antibody) selection might be driving the apparent positive selection, analogous to the antigenic drift observed in the influenza virus hemagglutinin (HA). However, careful antigenic and genetic comparison of the G glycoprotein does not provide evidence of antigenic drift in the G molecule, in agreement with recently published data which did not indicate antigenic drift in the G protein with human sera. Alternative explanations to the immune-driven selection hypothesis are offered to account for the high level of G-protein genetic diversity highlighted in this study.