Fine Mapping and Characterization of the L-Polymerase-Binding Domain of the Respiratory Syncytial Virus Phosphoprotein

Abstract

The minimum requirement for an active RNA-dependent RNA polymerase of respiratory syncytial virus (RSV) is a complex made of two viral proteins, the polymerase large protein (L) and the phosphoprotein (P). Here we have investigated the domain on P that is responsible for this critical P-L interaction. 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. The role of this molecular recognition element of about 30 amino acid residues in the L-P interaction and RNA polymerase activity was evaluated in cellula using an RSV minigenome system and site-directed mutagenesis. The results highlighted the critical role of hydrophobic residues located in this region. IMPORTANCERespiratory syncytial 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 responsible for this L-P interaction and the formation of an active L-P complex. These findings extend 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.H uman respiratory syncytial 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 increasingly 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 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 infection. Protein-protein interactions are potential targets for antiviral chemotherapy (4). The viral RNA-dependent RNA polymerase (RdRp) complex represents an attractive 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 facilitat...

New Insights into Structural Disorder in Human Respiratory Syncytial Virus Phosphoprotein and Implications for Binding of Protein Partners

“…6B), and it was proposed that this region might fold into a helix (30). Under our experimental conditions this region did not display any significant SSP, conformational exchange, or internal contacts, similarly to P NCBD .…”

“…The location of RdRp protein binding sites is also shown, for regions with and without significant SSPs. (13,24,25,30) and C terminally His-tagged hRSV nucleoprotein (13), N NTD (N residues 31-252) (14), and the K170A/R185A N mono mutant (24). Expression and Purification of Proteins-All proteins were expressed in E. coli BL21(DE3).…”

“…15 N-and 15 N, 13 C-labeled P protein samples for NMR experiments were produced in minimum M9 medium supplemented with 1 g liter Ϫ1 15 NH 4 Cl (Eurisotop), 4 or 3 g liter Ϫ1 unlabeled or 13 C-labeled glucose (Eurisotop), and 100 g ml Ϫ1 of ampicillin, using a protocol adapted from cultures in rich medium (30). Bacteria were disrupted (Constant Systems Ltd) in 50 mM Tris, pH 7.8, 60 mM NaCl, 1 mM EDTA, 2 mM ␤-mercaptoethanol, 0.2% Triton X-100 lysis buffer.…”

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases

“…However, P and L from PIV5 (75) and SeV (7) individually produced in mammalian cells cannot associate. Likewise, the RSV L protein cannot be detected in the absence of P even when overproduced in insect cells (18). Finally, even VSV L produced alone is rather unstable (16,17) and largely stabilized by P with a similar transient requirement for HSP90 activity (Fig.…”

“…How P and L from members of the order Mononegavirales associate into a functional polymerase is not well understood. On the one hand, L is stabilized by P, as shown for vesicular stomatitis virus (VSV) (16,17), Sendai virus (7), respiratory syncytial virus (RSV) (18), Ebola virus (EboV) (19), and measles virus (MeV) (20). In the case of VSV, P binding induces prominent conformational changes in L (21), the atomic structure of which, in complex with a P fragment, has been very recently solved by cryoelectron microscopy (22).…”