Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P interaction using a two-hybrid system

Mallipeddi, Sanjay K.; Lupiani, Blanca; Samal, Siba K.

doi:10.1099/0022-1317-77-5-1019

Cited by 27 publications

(25 citation statements)

References 21 publications

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“…However, we cannot exclude the possibility that the P NTD binding site partially overlaps the P CTD binding site on N. Furthermore, although the N NTD is a globular, alpha-helical domain that should not undergo major conformational changes between N 0 and N-RNA forms, P NTD binding on N mono could induce a local conformational change that renders the P CTD binding site inaccessible. In any case, our results concerning the RSV N 0 -P NTD interaction are in agreement with data obtained previously with BRSV showing a direct interaction between P NTD (residues 1 to 40) and N using the yeast two-hybrid system (20). Since P and N are highly conserved between BRSV and HRSV, it is likely that P NTD is the main N 0 binding site for both viruses.…”

Section: Discussionsupporting

confidence: 82%

“…3E). Our data showed that N mono copurified with GST-P and GST-P but not with GST-P [1][2][3][4][5][6][7][8][9][10], GST-P [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], GST-P , or GST-P . These results revealed that an N mono -specific binding site is located between amino acids 1 and 29 of P. It is noteworthy that residues 1 to 10, if not sufficient to interact with N mono , are required for the interaction.…”

Section: Resultsmentioning

confidence: 99%

“…The C-terminal region of P (aa 231 to 241) is involved in the interaction with the NC (12,18). Some experiments performed with the close homolog bovine RSV (BRSV) P protein have suggested that the aa 161 to 180 region could also participate in N-P interactions (19) and that the N terminus of P (residues 1 to 40) interacts with N (20).…”

mentioning

confidence: 99%

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

Galloux

Gabiane

Sourimant

et al. 2015

J Virol

113

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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...

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Section: Discussionsupporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

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

Galloux

Gabiane

Sourimant

et al. 2015

J Virol

113

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“…The N protein is present in large amounts in the virion and in infected cells and has several functions. In combination with P, L and possibly M2-2, the N protein is a major element of the nucleocapsid and protects the viral genome RNA from RNAse [93,112]. The N protein seems to play a role in the transition between the transcription and the replication phases of the viral RNA [93].…”

Section: Nucleocapsid Proteinsmentioning

confidence: 99%

“…In combination with P, L and possibly M2-2, the N protein is a major element of the nucleocapsid and protects the viral genome RNA from RNAse [93,112]. The N protein seems to play a role in the transition between the transcription and the replication phases of the viral RNA [93]. The P protein has 241 AA [2] and appears to act as a chaperone for soluble N and is implicated as a regulation factor for viral transcription and replication.…”

Section: Nucleocapsid Proteinsmentioning

confidence: 99%

Bovine respiratory syncytial virus infection

Valarcher¹,

2007

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-Bovine respiratory syncytial virus (BRSV) belongs to the pneumovirus genus within the family Paramyxoviridae and is a major cause of respiratory disease in young calves. BRSV is enveloped and contains a negative sense, single-stranded RNA genome encoding 11 proteins. The virus replicates predominantly in ciliated respiratory epithelial cells but also in type II pneumocytes. It appears to cause little or no cytopathology in ciliated epithelial cell cultures in vitro, suggesting that much of the pathology is due to the host's response to virus infection. RSV infection induces an array of pro-inflammatory chemokines and cytokines that recruit neutrophils, macrophages and lymphocytes to the respiratory tract resulting in respiratory disease. Although the mechanisms responsible for induction of these chemokines and cytokines are unclear, studies on the closely related human (H)RSV suggest that activation of NF-κB via TLR4 and TLR3 signalling pathways is involved. An understanding of the mechanisms by which BRSV is able to establish infection and induce an inflammatory response has been facilitated by advances in reverse genetics, which have enabled manipulation of the virus genome. These studies have demonstrated an important role for the non-structural proteins in anti-interferon activity, a role for a virokinin, released during proteolytic cleavage of the fusion protein, in the inflammatory response and a role for the SH and the secreted form of the G protein in establishing pulmonary infection. Knowledge gained from these studies has also provided the opportunity to develop safe, stable, live attenuated virus vaccine candidates.

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Mapping the phosphoprotein binding site on Sendai virus NP protein assembled into nucleocapsids

et al. 2004

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To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.

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Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N-P interaction using a two-hybrid system

Cited by 27 publications

References 21 publications

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

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

Bovine respiratory syncytial virus infection

Mapping the phosphoprotein binding site on Sendai virus NP protein assembled into nucleocapsids

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