TopoBuilding precision vaccines: To induce strong and targeted neutralizing antibody (nAb) responses against vaccine targets, one strategy has been to use computationally designed immunogens. However, the structural complexity of many known neutralization epitopes has posed a major challenge for the design of accurate epitope mimetics. Sesterhenn et al. created a protein design algorithm called TopoBuilder to design scaffolds for irregular and discontinuous neutralization epitopes. As a proof of principle, the authors generated epitope-focused immunogens based on the prefusion conformation of the respiratory syncytial virus (RSV) fusion protein. When these immunogens were used to vaccinate mice and nonhuman primates in RSV infection models, they generated targeted nAb responses to RSV and boosted site-specific nAb responses in heterologous prime-boost vaccination schemes. Science , this issue p. eaay5051

Significance: Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children; however, no vaccine exists and current immunoprophylaxis regimes are both expensive and incompletely protective. We report the crystal structure of the HRSV M2-1 transcription factor that is essential for virus gene expression and thus growth. This structure reveals how M2-1 forms an extremely stable tetramer and has allowed us to pinpoint the location of critical regions that regulate M2-1 activity, providing insight into its function. This structure may represent a potent target for new antiviral compounds.

Respiratory syncytial virus (RSV) RNA synthesis occurs in cytoplasmic inclusion bodies (IBs) in which all the components of the viral RNA polymerase are concentrated. In this work, we show that RSV P protein recruits the essential RSV transcription factor M2-1 to IBs independently of the phosphorylation state of M2-1. We also show that M2-1 dephosphorylation is achieved by a complex formed between P and the cellular phosphatase PP1. We identified the PP1 binding site of P, which is an RVxF-like motif located nearby and upstream of the M2-1 binding region. NMR confirmed both P-M2-1 and P-PP1 interaction regions in P. When the P–PP1 interaction was disrupted, M2-1 remained phosphorylated and viral transcription was impaired, showing that M2-1 dephosphorylation is required, in a cyclic manner, for efficient viral transcription. IBs contain substructures called inclusion bodies associated granules (IBAGs), where M2-1 and neo-synthesized viral mRNAs concentrate. Disruption of the P–PP1 interaction was correlated with M2-1 exclusion from IBAGs, indicating that only dephosphorylated M2-1 is competent for viral mRNA binding and hence for a previously proposed post-transcriptional function. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no potent antivirals are available against RSV, it is essential to better understand the mechanisms of viral replication to develop new antiviral strategies. Here we have investigated the mechanisms by which two essential components of the viral RNA polymerase machinery, the phosphoprotein P and the M2-1 transcription factor, interact and function. We identified the amino acid residues of P critical for this interaction and showed that they are required for P recruiting M2-1 to cytoplasmic inclusions, where viral polymerase complex proteins concentrate and viral RNA synthesis occurs. We also showed that M2-1 dephosphorylation, required for viral transcription, is achieved by a complex formed between P and the cellular phosphatase PP1. The region of P binding to PP1 is located nearby and upstream of the M2-1 binding domain. This is the first report showing that the phosphoprotein of a negative strand RNA virus can hijack a cellular phosphatase to modulate the phosphorylation state of its partners. These two P regions interacting with M2-1 and PP1 are also potential targets for future antiviral therapy.

The RNA genome of respiratory syncytial virus (RSV) is constitutively encapsidated by the viral nucleoprotein N, thus forming a helical nucleocapsid. Polymerization of N along the genomic and antigenomic RNAs is concomitant to replication and requires the preservation of an unassembled monomeric nucleoprotein pool. To this end, and by analogy with Paramyxoviridae and Rhabdoviridae , it is expected that the viral phosphoprotein P acts as a chaperone protein, forming a soluble complex with the RNA-free form of N (N ⁰ -P complex). Here, we have engineered a mutant form of N that is monomeric, is unable to bind RNA, still interacts with P, and could thus mimic the N ⁰ monomer. We used this N mutant, designated N ^mono , as a substitute for N ⁰ in order to characterize the P regions involved in the N ⁰ -P complex formation. Using a series of P fragments, we determined by glutathione S -transferase (GST) pulldown assays that the N and C termini of P are able to interact with N ^mono . We analyzed the functional role of amino-terminal residues of P by site-directed mutagenesis, using an RSV polymerase activity assay based on a human RSV minireplicon, and found that several residues were critical for viral RNA synthesis. Using GST pulldown and surface plasmon resonance assays, we showed that these critical residues are involved in the interaction between P[1-40] peptide and N ^mono in vitro . Finally, we showed that overexpression of the peptide P[1-29] can inhibit the polymerase activity in the context of the RSV minireplicon, thus demonstrating that targeting the N ⁰ -P interaction could constitute a potential antiviral strategy. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine or efficient antiviral treatment is available against RSV, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. RSV phosphoprotein P, the main RNA polymerase cofactor, is believed to function as a chaperon protein, maintaining N as a nonassembled, RNA-free protein (N ⁰ ) competent for RNA encapsidation. In this paper, we provide the first evidence, to our knowledge, that the N terminus of P contains a domain that binds specifically to this RNA-free form of N. We further show that overexpression of a small peptide spanning this region of P can inhibit viral RNA synthesis. These findings extend our understanding of the function of RSV RNA polymerase and point to a new target for the development of drugs against this virus.

PB1-F2 is a small accessory protein encoded by an alternative open reading frame in PB1 segments of most influenza A virus. PB1-F2 is involved in virulence by inducing mitochondria mediated immune cells apoptosis, increasing inflammation, and enhancing predisposition to secondary bacterial infections. Using biophysical approaches we characterized membrane disruptive activity of the full-length PB1-F2 (90aa), its N-terminal domain (52aa), expressed by currently circulating H1N1 viruses, and its C-terminal domain (38aa). Both full-length and N-terminal domain of PB1-F2 are soluble at pHs ≤ 6, while the C-terminal fragment was found soluble only at pH ≤ 3. All three peptides are intrinsically disordered. At pH ≥7, the C-terminal part of PB1-F2 spontaneously switches to amyloid oligomers while full-length and N-terminal domain of PB1-F2 aggregate to amorphous structures. When incubated with anionic liposomes at pH 5, full-length and C-terminal part of PB1-F2 assemble into amyloid structures and disrupt membrane at nM concentrations. PB1-F2 and its C-terminal exhibit no significant antimicrobial activity. When added in the culture medium of mammalian cells, PB1-F2 amorphous aggregates show no cytotoxicity, while PB1-F2 pre-assembled into amyloid oligomers or fragmented nanoscaled fibrils was highly cytotoxic. Furthermore, the formation of PB1-F2 amyloid oligormers in infected cells was directly reflected by membrane disruption and cell death as observed in U937 and A549 cells. Altogether our results demonstrate that membrane-lytic activity of PB1-F2 is closely linked to supramolecular organization of the protein of the targeted membranes.

Measurements of the presence of prions in biological tissues or fluids rely more and more on cell-free assays. Although protein misfolding cyclic amplification (PMCA) has emerged as a valuable, sensitive tool, it is currently hampered by its lack of robustness and rapidity for high-throughput purposes. Here, we made a number of improvements making it possible to amplify the maximum levels of scrapie prions in a single 48-h round and in a microplate format. The amplification rates and the infectious titer of the PMCA-formed prions appeared similar to those derived from the in vivo laboratory bioassays. This enhanced technique also amplified efficiently prions from different species, including those responsible for human variant Creutzfeldt-Jakob disease. This new format should help in developing ultrasensitive, high-throughput prion assays for cognitive, diagnostic, and therapeutic applications. IMPORTANCE The method developed here allows large-scale, fast, and reliable cell-free amplification of subinfectious levels of prions from different species. The sensitivity and rapidity achieved approach or equal those of other recently developed prion-seeded conversion assays. Our simplified assay may be amenable to high-throughput, automated purposes and serve in a complementary manner with other recently developed assays for urgently needed antemortem diagnostic tests, by using bodily fluids containing small amounts of prion infectivity. Such a combination of assays is of paramount importance to reduce the transfusion risk in the human population and to identify asymptomatic carriers of variant Creutzfeldt-Jakob disease.