SummaryIn sporulating Bacillus, major processes like virulence gene expression and sporulation are regulated by communication systems involving signalling peptides and regulators of the RNPP family. We investigated the role of one such regulator, NprR, in bacteria of the Bacillus cereus group. We show that NprR is a transcriptional regulator whose activity depends on the NprX signalling peptide. In association with NprX, NprR activates the transcription of an extracellular protease gene (nprA) during the first stage of the sporulation process. The transcription start site of the nprA gene has been identified and the minimal region necessary for full activation has been characterized by promoter mutagenesis. We demonstrate that the NprX peptide is secreted, processed and then reimported within the bacterial cell. Once inside the cell, the mature form of NprX, presumably the SKPDIVG heptapeptide, directly binds to NprR allowing nprA transcription. Alignment of available NprR sequences from different species of the B. cereus group defines seven NprR clusters associated with seven NprX heptapeptide classes. This cell-cell communication system was found to be strain-specific with a possible cross-talk between some pherotypes. The phylogenic relationship between NprR and NprX suggests a coevolution of the regulatory protein and its signalling peptide.

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 M2-158–177 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 M2-158–177, 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.

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. IMPORTANCERespiratory 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. T he human respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (1). HRSV infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. It is also recognized as a significant cause of severe respiratory infections in the elderly. The virus belongs to the Mononegavirales order and constitutes the prototype virus of the Pneumovirus genus of the Paramyxoviridae family. As f...

In Gram-positive bacteria, cell-to-cell communication mainly relies on extracellular signaling peptides, which elicit a response either indirectly, by triggering a two-component phosphorelay, or directly, by binding to cytoplasmic effectors. The latter comprise the RNPP family (Rgg and original regulators Rap, NprR, PrgX and PlcR), whose members regulate important bacterial processes such as sporulation, conjugation, and virulence. RNPP proteins are increasingly considered as interesting targets for the development of new antibacterial agents. These proteins are characterized by a TPR-type peptide-binding domain, and except for Rap proteins, also contain an N-terminal HTH-type DNA-binding domain and display a transcriptional activity. Here, we elucidate the structure-function relationship of the transcription factor ComR, a new member of the RNPP family, which positively controls competence for natural DNA transformation in streptococci. ComR is directly activated by the binding of its associated pheromone XIP, the mature form of the comX/sigX-inducing-peptide ComS. The crystal structure analysis of ComR from Streptococcus thermophilus combined with a mutational analysis and in vivo assays allows us to propose an original molecular mechanism of the ComR regulation mode. XIP-binding induces release of the sequestered HTH domain and ComR dimerization to allow DNA binding. Importantly, we bring evidence that this activation mechanism is conserved and specific to ComR orthologues, demonstrating that ComR is not an Rgg protein as initially proposed, but instead constitutes a new member of the RNPP family. In addition, identification of XIP and ComR residues important for competence activation constitutes a crucial step towards the design of antagonistic strategies to control gene exchanges among streptococci.