Infection of host cells by the respiratory syncytial virus (RSV) is characterized by the formation of spherical cytoplasmic inclusion bodies (IBs). These structures, which concentrate all the proteins of the polymerase complex as well as some cellular proteins, were initially considered aggresomes formed by viral dead-end products. However, recent studies revealed that IBs are viral factories where viral RNA synthesis, i.e., replication and transcription, occurs. The analysis of IBs by electron microscopy revealed that they are membrane-less structures, and accumulated data on their structure, organization, and kinetics of formation revealed that IBs share the characteristics of cellular organelles, such as P-bodies or stress granules, suggesting that their morphogenesis depends on a liquid-liquid phase separation mechanism. It was previously shown that expression of the RSV nucleoprotein N and phosphoprotein P of the polymerase complex is sufficient to induce the formation of pseudo-IBs. Here, using a series of truncated P proteins, we identified the domains of P required for IB formation and show that the oligomeric state of N, provided it can interact with RNA, is critical for their morphogenesis. We also show that pseudo-IBs can form in vitro when recombinant N and P proteins are mixed. Finally, using fluorescence recovery after photobleaching approaches, we reveal that in cellula and in vitro IBs are liquid organelles. Our results strongly support the liquid-liquid phase separation nature of IBs and pave the way for further characterization of their dynamics. IMPORTANCE Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, elderly, and immunocompromised people. No vaccine or efficient antiviral treatment is available against this virus. The replication and transcription steps of the viral genome are appealing mechanisms to target for the development of new antiviral strategies. These activities take place within cytoplasmic inclusion bodies (IBs) that assemble during infection. Although expression of both the viral nucleoprotein (N) and phosphoprotein (P) allows induction of the formation of these IBs, the mechanism sustaining their assembly remains poorly characterized. Here, we identified key elements of N and P required for the scaffolding of IBs and managed for the first time to reconstitute RSV pseudo-IBs in vitro by coincubating recombinant N and P proteins. Our results provide strong evidence that the biogenesis of RSV IBs occurs through liquid-liquid phase transition mediated by N-P interactions.

Biomolecular condensates have emerged as an important subcellular organizing principle 1 . Replication of many viruses, including human respiratory syncytial virus (RSV), occurs in virus-induced compartments called inclusion bodies (IBs) or viroplasm 2,3 . IBs of negative-strand RNA viruses were recently shown to be biomolecular condensates that form through phase separation 4,5 . Here we report that the steroidal alkaloid cyclopamine and its chemical analogue A3E inhibit RSV replication by disorganizing and hardening IB condensates. The actions of cyclopamine and A3E were blocked by a point mutation in the RSV transcription factor M2-1. IB disorganization occurred within minutes, which suggests that these molecules directly act on the liquid properties of the IBs. A3E and cyclopamine inhibit RSV in the lungs of infected mice and are condensate-targeting drug-like small molecules that have in vivo activity. Our data show that condensate-hardening drugs may enable the pharmacological modulation of not only many previously undruggable targets in viral replication but also transcription factors at cancer-driving super-enhancers 6 .RSV is a major cause of respiratory illness in young children, the older people and individuals who are immunocompromised worldwide 7,8 . Currently, multiple targets are pursued for the development of a safe and effective therapy to treat RSV infections 9 .In infected cells, RSV induces the formation of cytoplasmic IBs, in which nucleoprotein (N), phosphoprotein (P), polymerase L, the transcription factor M2-1 and viral genomic RNA are concentrated. We recently demonstrated that IBs are 'viral factories' in which viral RNA synthesis occurs 3 . The morphology of IBs suggests that they are condensates formed by liquid-liquid phase separation (LLPS). A recent study showed that N and P were sufficient to drive the formation of pseudo-IB condensates through LLPS in vitro, both in cells and in biochemical assays 10 . However, these N-P pseudo-IB condensates are not functional, as they do not shelter RNA synthesis and do not reflect the complexity of IBs in virus-infected cells, which have multiple compartments. Strikingly similar in size and phase organization to the nucleolus condensate 11 , RSV IBs are multiphasic and contain a sub-compartment called the IB-associated granule (IBAG), which is composed of newly synthesized viral mRNA and M2-1 3,12 . Condensates have emerged as an important subcellular organizing principle 1 . An important question in anti-viral drug developmentand medicinal chemistry more generally-is whether these condensates are druggable. In principle, a drug that dissolved or hardened would prevent viral replication. Neither mechanism has yet been reported. Chemical analogues without hedgehog antagonismWe previously identified the hedgehog (HH) pathway antagonist cyclopamine (CPM) as a potent inhibitor of RSV replication 13 . Inhibition of Sonic hedgehog (SHH) signalling is an unwanted feature of CPM as an RSV inhibitor. On the basis of the binding model of the Smoothe...

Rates of spontaneous mutation determine the ability of viruses to evolve, infect new hosts, evade immunity and undergo drug resistance. Contrarily to RNA viruses, few mutation rate estimates have been obtained for DNA viruses, because their high replication fidelity implies that new mutations typically fall below the detection limits of Sanger and standard next-generation sequencing. Here, we have used a recently developed high-fidelity deep sequencing technique (Duplex Sequencing) to score spontaneous mutations in human adenovirus 5 under conditions of minimal selection. Based on >200 single-base spontaneous mutations detected throughout the entire viral genome, we infer an average mutation rate of 1.3 × 10−7 per base per cell infection cycle. This value is similar to those of other, large double-stranded DNA viruses, but an order of magnitude lower than those of single-stranded DNA viruses, consistent with the possible action of post-replicative repair. Although the mutation rate did not vary strongly along the adenovirus genome, we found several sources of mutation rate heterogeneity. First, two regions mapping to transcription units L3 and E1B-IVa2 were significantly depleted for mutations. Second, several point insertions/deletions located within low-complexity sequence contexts appeared recurrently, suggesting mutational hotspots. Third, mutation probability increased at GpC dinucleotides. Our findings suggest that host factors may influence the distribution of spontaneous mutations in human adenoviruses and potentially other nuclear DNA viruses.

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It is now well known that hemostasis is directly involved in the benefits induced by physical activity. It has recently been shown that the baseline mean platelet volume (MPV) may be a predictor of endurance performance. We aimed to explore whether platelet parameters are associated with VO2max as well as running duration and speed in a short-duration exhaustive exercise test. Thirty healthy male subjects (10 sedentary and 20 trained) performed an incremental running test until exhaustion. MPV, platelet distribution width (PDW), platelet (Plt) count, and plateletcrit (Pct) were determined before exercise, immediately after exercise and after 30' recovery. Training status did not produce any difference in the baseline levels or in the post-exercise increases found in all the parameters tested. VO2max, test duration, and running speed were not correlated with any baseline parameter. Although MPV was found to be a predictor of endurance performance in long-duration exercise, the results of the present study are consistent with the hypothesis that MPV may not be a significant marker of performance in short-duration exhaustive exercise. Likewise, more research is needed to ascertain whether platelet activation is a reliable performance predictor in other exercise settings.

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Most DNA viruses exhibit relatively low rates of spontaneous mutation. However, the molecular mechanisms underlying DNA virus genetic stability remain unclear. In principle, mutation rates should not depend solely on polymerase fidelity, but also on factors such as DNA damage and repair efficiency. Most eukaryotic DNA viruses interact with the cellular DNA damage response (DDR), but the role of DDR pathways in preventing mutations in the virus has not been tested empirically. To address this goal, we serially transferred human adenovirus type 5 in cells in which the telangiectasia-mutated PI3K-related protein kinase (ATM), the ATM/Rad3-related (ATR) kinase, and the DNA-dependent protein kinase (DNA-PK) were chemically inactivated, as well as in control cells displaying normal DDR pathway functioning. High-fidelity deep sequencing of these viral populations revealed mutation frequencies in the order of one-millionth, with no detectable effect of the inactivation of DDR mediators ATM, ATR, and DNA-PK on adenovirus sequence variability. This suggests that these DDR pathways do not play a major role in determining adenovirus genetic diversity.

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