Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex

Asenjo, Ana B.; Calvo, Enrique; Villanueva, Nieves

doi:10.1099/vir.0.82165-0

Cited by 48 publications

(47 citation statements)

References 21 publications

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“…S2A) whereas the C-terminal face also shows extensive positive charge separated by four "tracts" of negative charge. The extensive positive charge of the tetramer surface is consistent with the high pI and previously identified interactions with negatively charged ligands, namely RNA and P protein (7)(8)(9). The putative RNA binding surface is entirely populated with charged residues that would favor electrostatic interactions with the negatively charged RNA phosphate backbone.…”

Section: Resultssupporting

confidence: 53%

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Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Tanner

Ariza

Richard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

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The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.H uman respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract illness in young children and the immunocompromised. HRSV is a pneumovirus of the Paramyxoviridae family of the order Mononegavirales-the nonsegmented negative-strand RNA viruses. Its genome encodes 10 genes that are each transcribed by an RNA-dependant RNA polymerase (RdRp) into single mRNAs. During transcription, the RdRp uses a single promoter in the 3′ leader region (Le) of the genome (1) and responds to gene start and gene end sequences flanking each gene, directing initiation and termination of mRNA transcription, respectively (2). During genome replication, the RdRp bypasses these signals to synthesize a full-length antigenome. The virus-encoded components needed for RNA replication are the large protein (L), the nucleocapsid protein (N), and the phosphoprotein (P). However, complete transcription of mRNAs also requires the M2-1 transcription antiterminator protein (3, 4).M2-1 prevents premature transcription termination both intra-and intergenically (5, 6). M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7, 8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11). Based on mutational analysis and a partial M2-1 structure determined using NMR (12, 13), M2-1 is predicted to comprise four functionally significant regions: an N-terminal Cys 3 -His 1 zinc-binding domain (ZBD) (14); an alpha-helical region proposed to mediate oligomerization (11); the "core" domain (residues ∼58-177) assigned to RNA-and P-binding; and an unstructured C terminus. The core exh...

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Section: Resultssupporting

confidence: 53%

“…M2-1 is essential for HRSV multiplication although it is not currently known how M2-1 effects its role, and deciphering this role is complicated by its multiple interactions with other viral components, namely P (7,8), RNA (9), and the matrix protein (M) (10). M2-1 is a 194 amino acid, basic protein that forms a stable tetramer in solution (11).…”

mentioning

confidence: 99%

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Tanner

Ariza

Richard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

131

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“…The P protein has been shown to present multiple sites of phosphorylation, at threonine residues 46 (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, the major phosphorylation sites of P are dispensable for RSV replication in vitro (24), and the exact role of phosphorylation in P activity is still debated.…”

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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“…Besides critical involvement in the formation of IBs and in the hRSV polymerase complex, the roles of the N and P proteins in particle morphogenesis are not clear. However, a role for P and P-M interaction in virus entry, and potentially virus assembly, was recently proposed (48,49).…”

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confidence: 99%

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

Meshram

Baviskar

Ognibene

et al. 2016

J Virol

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Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCEhRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines. H uman respiratory syncytial virus (hRSV) is an important viral agent of respiratory tract disease in infants, children, immunosuppressed individuals, and the elderly (1-3). In pursuit of a vaccine to prevent hRSV disease, a virus-like particle (...

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Phosphorylation of human respiratory syncytial virus P protein at threonine 108 controls its interaction with the M2-1 protein in the viral RNA polymerase complex

Cited by 48 publications

References 21 publications

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation

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

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

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