Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Simabuco, Fernando Moreira; Asara, John M.; Guerrero, Manuel Ceballos; Libermann, Towia A.; Zerbini, Luiz Fernando; Ventura, Armando Morais

doi:10.1590/s1517-83822011000100043

Cited by 16 publications

(16 citation statements)

References 19 publications

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“…The only structured part appears to be the tetramerization domain, which has been investigated by bioinformatic tools (26), resistance to protease digestion (28,41), and deletion series (13,25,42). Although fragment Y*, which exhibits high stability and homogeneity (25,27,28), has been acknowledged as the core of the tetramerization domain, the OD of hRSV P is often represented by fragment X (Table 1), longer then Y* by 15 residues at its N terminus.…”

Section: Discussionmentioning

confidence: 99%

“…Bioinformatic and biochemical investigations have established that hRSV P is tetrameric and contains large disordered N-and C-terminal regions (25)(26)(27). Fragment Y* (Table 1) was described as a minimal oligomerization domain (OD) with predicted helical coiled-coil structure (28).…”

Section: Human Respiratory Syncytial Virus (Hrsv)mentioning

confidence: 99%

“…P also acts as a chaperone for neo-synthesized N by forming an N 0 ⅐P complex that preserves N in a monomeric and RNA-free state (23). We have shown previously that formation of hRSV NC⅐P and N 0 ⅐P complexes proceeds via two distinct binding sites on P (14, 24).Bioinformatic and biochemical investigations have established that hRSV P is tetrameric and contains large disordered N-and C-terminal regions (25)(26)(27). Fragment Y* (Table 1) was described as a minimal oligomerization domain (OD) with predicted helical coiled-coil structure (28).…”

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confidence: 99%

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

Pereira¹,

Cardone²,

Lassoued³

et al. 2017

Journal of Biological Chemistry

116

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Edited by Charles E. SamuelPhosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales. It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that Nand C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase. Human respiratory syncytial virus (hRSV),3 a member of the family Pneumoviridae (1) and order Mononegavirales (MNV), is the main viral cause of lower respiratory tract illness worldwide, and the main agent responsible for bronchiolitis and pneumonia in infants (2). All children have been infected by the age of two, requiring hospitalization in ϳ5% cases (3). Elderly and immunocompromised adults are also at increased risk. No efficient treatment is presently available for hRSV (4), and vaccination is challenging due to complex immunogenicity (5). The search for hRSV antiviral drugs directed toward specific viral functions is therefore still ongoing (6).The hRSV RNA-dependent RNA complex (RdRp) constitutes a virus-specific target with specific protein-protein interactions that have not all been investigated in detail (7). It uses the nonsegmented single-stranded negative sense RNA genome of hRSV as a template. In infected cells, the viral RdRp is found in specific inclusion bodies (8), which have been shown to be transcription and replication centers for other Mononegavirales, e.g. rabies (9) and vesicular stomatitis viruses (10). The apo RdRp complex is composed a minima of the large catalytic subunit (L) and its essential cofactor, the phosphoprotein (P) (11, 12). The P protein plays a central role in the RdRp by interacting with all main RdRp components. During transcription and replication it tethers the L protein to the nucleocapsid (NC), consisting of the genomic RNA packaged by the nucleoprotein (N), by direct interaction with N (13-16). hRSV P also binds to the transcription antitermination factor M2-1 (17-19). Phosphorylation of P has been proposed to regulate these interactions, although it is not essential for replication (20 -22). P also acts as a chaperone for neo-synthesized N by forming an N 0 ⅐P complex that preserves N in a monomeric and RNA-free state (23). We have shown previously that formation of hRSV NC⅐P and ...

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Section: Discussionmentioning

confidence: 99%

Section: Human Respiratory Syncytial Virus (Hrsv)mentioning

confidence: 99%

mentioning

confidence: 99%

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

Pereira¹,

Cardone²,

Lassoued³

et al. 2017

Journal of Biological Chemistry

116

View full text Add to dashboard Cite

show abstract

“…The RSV P protein forms highly stable tetramers and can be divided into three domains: an N-terminal domain (P NTD , comprising residues 1 to ϳ120), a central oligomerization domain (P OD , comprising residues ϳ120 to 160), and a C-terminal domain (P CTD , comprising residues 161 to 241) (15,22,27,28). P NTD and P CTD 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 the last 9 C-terminal residues of P CTD 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).…”

mentioning

confidence: 99%

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

Sourimant

Rameix‐Welti

Gaillard

et al. 2015

J Virol

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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...

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“…Sua função é fazer com que a proteína N se ligue ao RNA viral (Ruigrok, Crepin e Kolakofsy, 2011), e exceto pela sua região central, a proteína P é fracamente estruturada. Os resíduos do N-terminal 1-103 e do C-terminal 200-241 formam regiões intrinsecamente desordenadas (Castagne et al, 2004;Simabuco et. al., 2011).…”

Section: Interação Entre a Proteína N E Punclassified

Caracterização da nucleoproteína e da fosfoproteína do vírus respiratório sincicial humano quanto a suas propriedades imunogênicas e de interação com proteínas celulares.

Oliveira¹

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Structural analysis of human respiratory syncytial virus P protein: identification of intrinsically disordered domains

Cited by 16 publications

References 19 publications

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

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

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

Caracterização da nucleoproteína e da fosfoproteína do vírus respiratório sincicial humano quanto a suas propriedades imunogênicas e de interação com proteínas celulares.

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