Protein interaction domains of the measles virus nucleocapsid protein (NP)

Measles virus nucleoprotein is the most abundant viral protein and tightly encapsidates viral genomic RNA to support viral transcription and replication. Major phosphorylation sites of nucleoprotein include the serine residues at locations 479 and 510. Minor phosphorylation residues have yet to be identified, and their functions are poorly understood. In our present study, we identified nine putative phosphorylation sites by mass spectrometry and demonstrated that threonine residue 279 (T279) is functionally significant. Minigenome expression assays revealed that a mutation at the T279 site caused a loss of activity. Limited proteolysis and electron microscopy suggested that a T279A mutant lacked the ability to encapsidate viral RNA but was not denatured. Furthermore, dephosphorylation of the T279 site by alkaline phosphatase treatment caused deficiencies in nucleocapsid formation. Taken together, these results indicate that phosphorylation at T279 is a prerequisite for successful nucleocapsid formation. Measles virus (MV), a member of the Morbillivirus genus in the Paramyxoviridae family, is an important human pathogen that causes disease characterized by fever, cough, coryza, conjunctivitis, and a maculopapular rash. Although the use of effective vaccines has decreased global mortality from measles, it remains a major cause of high mortality among children in developing countries (1-3). MV has a nonsegmented negativestranded RNA genome containing six structural genes encoding nucleoprotein (N), phosphoprotein (P), matrix (M) protein, fusion (F) protein, hemagglutinin (H) protein, and large (L) protein (4), and the P gene produces two accessory proteins, known as V and C (5, 6). N proteins encapsidate viral genomic RNA to support viral transcription and replication by an RNA-dependent RNA polymerase (RdRp) L protein. The P protein is a multifunctional protein (7,8) that assists with viral transcription and replication as a cofactor of the L protein (9, 10). The C and V accessory proteins suppress host immune responses (11,12). The M protein helps virus assembly, and the F and H proteins are required for membrane fusion and binding to the host cellular receptor, respectively (13).N protein is the most abundant viral protein in infected cells (14) and is mainly required for viral transcription and replication. N proteins tightly associate with the viral genome and antigenome to form an N-RNA complex with a herringbone-like structure (15,16). N proteins associate with every 6 bases of the 15,894-nucleotide viral genome and fully cover the genome RNA (17). This tight encapsidation allows the viral genome to be resistant to RNases and small interfering RNAs (18,19). Viral transcription and replication occur on the N-RNA complex in association with viral RdRp (vRdRp), composed of L and P proteins. This complex of N-RNA, P protein, and L protein is called the nucleocapsid (NC) and comprises 2,649 copies of the N protein, about 300 copies of the P protein, and about 20 to 30 copies of the L protein (20)(21)(22). Singly...

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“…2D) and is known to be important for N-protein function (33,34). In a previous analysis (27) and the present MS analysis (Fig.…”

Section: Identification Of a Minor Phosphorylation Site Of MV N Proteinsupporting

confidence: 60%

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Sugai¹,

Sato²,

Hagiwara³

et al. 2014

J Virol

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“…We have shown that enhanced sensitivity to 1,25-(OH) 2 D 3 is an intrinsic property of OCL precursors from Paget's patients, and that it may result from increased affinity of the vitamin D receptor for 1,25-(OH) 2 D 3 (26). Similarly, OCL precursors transduced with the MVNP gene showed increased expression of 24-hydroxylase mRNA in response to 1,25-(OH) 2 (27) demonstrated that MVNP proteins can interact with a variety of cellular proteins. Immunoprecipitation studies using glutathione S-transferase nucleocapsid protein constructs demonstrated that MVNP interacted specifically with at least 12 cellular proteins.…”

Section: Discussionmentioning

confidence: 83%

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype

et al. 2000

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“…As discussed above, MV N TAIL is required for stable binding of N to P (18,20,26). To determine whether the isolated N TAIL domain was able to bind P, we tested the ability of bacterial lysates expressing hexahistidine tagged N TAIL to pull down untagged P onto an IMAC resin.…”

Section: Resultsmentioning

confidence: 99%

“…In Morbilliviruses and Respiroviruses, the region responsible for binding of nucleocapsids to P is located within N TAIL (13,18,20,24). Consequently, N TAIL is required for N⅐RNA to act as a template for viral RNA synthe-sis (13,16,25).…”

mentioning

confidence: 99%

The C-terminal Domain of the Measles Virus Nucleoprotein Is Intrinsically Disordered and Folds upon Binding to the C-terminal Moiety of the Phosphoprotein

Longhi

Receveur-Brechot

Karlin

et al. 2003

Journal of Biological Chemistry

268

324

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The nucleoprotein of measles virus consists of an Nterminal moiety, N CORE , resistant to proteolysis and a C-terminal moiety, N TAIL , hypersensitive to proteolysis and not visible as a distinct domain by electron microscopy. We report the bacterial expression, purification, and characterization of measles virus N TAIL . Using nuclear magnetic resonance, circular dichroism, gel filtration, dynamic light scattering, and small angle x-ray scattering, we show that N TAIL is not structured in solution. Its sequence and spectroscopic and hydrodynamic properties indicate that N TAIL belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The same epitopes are exposed in N TAIL and within the nucleoprotein, which rules out dramatic conformational changes in the isolated N TAIL domain compared with the full-length nucleoprotein. Most unstructured proteins undergo some degree of folding upon binding to their partners, a process termed "induced folding." We show that N TAIL is able to bind its physiological partner, the phosphoprotein, and that it undergoes such an unstructured-to-structured transition upon binding to the C-terminal moiety of the phosphoprotein. The presence of flexible regions at the surface of the viral nucleocapsid would enable plastic interactions with several partners, whereas the gain of structure arising from induced folding would lead to modulation of these interactions. These results contribute to the study of the emerging field of natively unfolded proteins.

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Protein interaction domains of the measles virus nucleocapsid protein (NP)

Cited by 54 publications

References 39 publications

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Newly Identified Minor Phosphorylation Site Threonine-279 of Measles Virus Nucleoprotein Is a Prerequisite for Nucleocapsid Formation

Osteoclasts expressing the measles virus nucleocapsid gene display a pagetic phenotype

The C-terminal Domain of the Measles Virus Nucleoprotein Is Intrinsically Disordered and Folds upon Binding to the C-terminal Moiety of the Phosphoprotein

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