By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site

By use of recombinant proteins and serial deletions, an L binding site was mapped in the C-terminal region of P, just upstream of the N-RNA binding site. site-directed mutagenesis. The results highlighted the crucial role of hydrophobic residues located in this region. IMPORTANCE Respiratory syncytial computer virus (RSV) is the leading cause of lower respiratory tract illness in infants. Since no vaccine and no good antivirals against RSV are available, it is essential to better understand how the viral machinery functions in order to develop new antiviral strategies. Like all negative-strand RNA viruses, RSV codes for its own machinery to replicate and transcribe its genome. The core of this machinery is composed of two proteins, the phosphoprotein (P) and the large protein (L). Here, using recombinant proteins, we have mapped and characterized the P domain name responsible for this L-P conversation and the formation Rabbit Polyclonal to EPS15 (phospho-Tyr849) of an active L-P complex. These findings lengthen our understanding of the mechanism of action of RSV RNA polymerase and allow us to define a new target for the development of drugs against RSV. INTRODUCTION Human respiratory syncytial computer virus (HRSV) is the leading cause of acute respiratory infections in infants worldwide and is the primary cause of infant hospitalization for respiratory infections (1). Moreover, RSV is progressively recognized as a significant cause of disease in the elderly population and can often be fatal for patients with compromised immune systems (2). In parallel with its human counterpart, bovine RSV (BRSV) constitutes a major cause of respiratory disease in calves, resulting in substantial economic losses to the cattle industry worldwide (3). Despite the substantial health and economic burden caused by RSV illness, there is currently no human vaccine or antiviral drug available. The only significant preventive treatment available is usually prophylaxis with palivizumab (Synagis), a humanized monoclonal antibody that has provided about 50% protection to high-risk children. Therefore, there is an urgent need to discover compounds capable of blocking RSV contamination. Protein-protein interactions are potential targets for antiviral chemotherapy (4). The viral RNA-dependent RNA polymerase (RdRp) complex represents a stylish target for drug discovery, because the different components have no cellular ortholog and are highly conserved between RSV strains. The mechanism of action of this complex involves highly specific and regulated protein-protein and RNA-protein interactions that we need to understand in order to facilitate drug design approaches. RSV belongs to the genus of the family, order (9, 10), considered to be viral factories where viral RNA Genipin synthesis takes place. Complete atomic structures of N and M2-1 are now available (11,C13). No atomic structure has been resolved, even partially, for P or L. However, the crystal structure of the human metapneumovirus (HMPV) P oligomerization domain name, formed by a tetrameric coiled-coil, has recently been resolved (14). Since the RSV and HMPV P proteins are highly comparable, with 78% identical residues between them, the RSV P oligomerization domain name should include at least residues 130 to 152, where a coiled-coil domain name is also predicted (15). The P protein is the main L cofactor and is essential for the formation of an active polymerase complex, allowing the L protein to gain access to the nucleocapsid, where the viral genome is usually sequestered; P interacts with both the L protein and the N protein simultaneously (16). The P protein has been shown to present multiple sites of phosphorylation, at threonine residues 46 and 108, serine residues 30, 39, 45, 54, 116, 117, 119, 156, 161, 232, and 237, and potentially also Ser86, Ser94, and Ser99 (17,C26). However, the major phosphorylation sites of P are Genipin dispensable for RSV replication (24), and the exact role of phosphorylation in P activity is still debated. The RSV P protein forms highly stable tetramers and can be divided into three domains: an N-terminal domain name (PNTD, comprising residues 1 to 120), a central oligomerization domain name (POD, comprising residues 120 to 160), and a C-terminal domain name (PCTD, comprising residues 161 to 241) (15, 22, 27, 28). PNTD and PCTD are predicted to be disordered regions (29), although some putative short -helices have been predicted between residues 14 and 25 and between residues 220 and 228 (27). Although it is now well established that this last 9 C-terminal residues of PCTD are critical for binding to N-RNA complexes (30, 31), a second region encompassing residues 161 to 180 could also be involved in N binding (32). However, the L-binding domain name Genipin of P is still debated. In this work, we have investigated the P-L interactions by using recombinant proteins. The RSV L protein was expressed using a baculovirus vector. This recombinant L protein was able to bind to recombinant P purified from bacteria. Serial deletions of P residues showed that the main L-binding domain name extends from residue 212 to residue 239 in Genipin the C-terminal.