Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich’s ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich’s ataxia therapies.

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-1_58–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-1_58–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1. Premature termination of transcription by the RNA-dependent RNA polymerase (RdRp) complex of respiratory syncytial virus (RSV) is prevented by the M2-1 protein. This transcription factor interacts with both RNA and viral phosphoprotein P, the main RdRp cofactor, through a specific “core” domain. Using NMR, we solved the 3D structure of this domain and characterized the surface residues involved in P- and RNA-binding. Based on these data, we designed point mutations impairing binding to either RNA or P. We studied the functional implications of these mutations for transcription and co-localization of full-length M2-1 in cytoplasmic inclusion bodies, where viral transcription likely occurs. We found that the RNA- and P-binding surfaces are in close proximity, accounting for competitive binding in vitro. Our results suggest that binding to both RNA and to P is necessary for transcriptional antitermination by M2-1, but that the role of the interaction with P is primarily to recruit M2-1 to the RdRp complex. Finally we show that the M2-1 core domain is homologous to the C-terminal domain of Ebola virus VP30 despite low sequence identity, solidifying the relationship between these two proteins and transcriptional regulation strategies shared by viruses belonging to the Filoviridae family and the Pneumovirinae subfamily.

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine–Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed ‘S1 proteins’. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their β-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4–5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1.

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.