C-Terminal phosphorylation of human respiratory syncytial virus P protein occurs mainly at serine residue 232

113

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...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Identification and Characterization of the Binding Site of the Respiratory Syncytial Virus Phosphoprotein to RNA-Free Nucleoprotein

Galloux

Gabiane

Sourimant

et al. 2015

113

“…Phosphorylation is mediated by the cellular casein kinase II (8, 33) on two clusters of serines: 116, 117, and 119 in the central region and 232 and 237 in the C-terminal region (26,27,31,33). Approximately 80% of P protein phosphorylation is localized to Ser-232, and the remaining 20% is distributed among Ser-116, -117, -119, and -237.…”

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confidence: 99%

The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

Lü¹,

Ma²,

Brazas³

et al. 2002

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33 P i -labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2-and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.The phosphoprotein (P protein) of human respiratory syncytial virus (RSV), a prototype Pneumovirus of the family Paramyxoviridae, is an essential component of the viral RNA polymerase, along with the large polymerase (L) and nucleocapsid (N) proteins (12,35). Interaction of the RSV P protein with the N and L proteins promotes the formation of a transcriptase complex that is essential for viral RNA transcription and replication (10,19,20). Although L protein is the catalytic RNA polymerase, P protein is essential for transcription and replication of viral RNA (7,14). In addition to the N, P, and L proteins, several viral proteins are required for RSV RNA synthesis. The antitermination function of M2-1 is essential for processive RNA synthesis and suppression of transcription termination in intergenic regions (6, 13). M2-2 has been postulated to have a role in regulating the switch between viral RNA transcription and replication processes (3, 17).The P protein of RSV subgroup A 2 is 241 amino acids in length, which is much shorter than the P proteins of other paramyxoviruses (5, 21), and forms homotetramers (1), similar to the Sendai virus P protein (29, 30). The interaction of the N and P proteins enables proper folding of N protein and enables N protein to encapsidate v...

“…In RSV P protein, two clusters of phosphorylation sites (amino acid residues 116, 117, and 119 and residues 232 and 237) have been identified (5,9,21,24,34). Phosphorylation of S232 was proposed to regulate RSV transcription; however, that study was based on in vitro assays (33).…”

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confidence: 99%

Identification of a Phosphorylation Site within the P Protein Important for mRNA Transcription and Growth of Parainfluenza Virus 5

Sun

Luthra

et al. 2011

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.Parainfluenza virus 5 (PIV5) is a prototypical member of the paramyxovirus family, which contains many human and animal pathogens, such as Sendai virus (SeV), mumps virus (MuV), respiratory syncytial virus (RSV), Hendra virus (HeV), and Nipah virus (NiV) (15). The negative-stranded RNA genome of PIV5 contains seven genes yet encodes eight proteins in the order NP-V/P-M-F-SH-HN-L (15). While V mRNA is transcribed faithfully by the V/P gene, P mRNA is produced by the V/P gene through an insertion of two nontemplate guanine residues at a specific sequence during viral RNA transcription (30). The phosphoprotein (P) and large polymerase (L) protein constitute the viral RNA-dependent RNA polymerase complex (vRdRP). The L protein has enzymatic activities capable of initiation, elongation, and termination of RNA synthesis, as well as addition of the 5Ј cap structure and 3Ј poly(A) sequence (15). The P protein does not have intrinsic enzymatic activity; however, it is an essential cofactor for the polymerase, regulating polymerase activity in viral RNA replication and transcription (15).The P proteins of all paramyxoviruses are heavily phosphorylated and hence are named phosphoproteins (15). However, the role of P protein phosphorylation in the replication of paramyxoviruses has been an enigma. It was initially suggested that phosphorylation of the P protein was essential for viral RNA synthesis. In paramyxovirus, the P proteins of SeV and RSV have been studied the most extensively. Up to 11 phosphorylation sites of SeV P protein were detected (14, 32), and S249 was identified as the major phosphorylation site (6, 7). However, mutation at S249 showed normal ...