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

Abstract

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...

Detection and Genome Analysis of a Lineage III Peste Des Petits Ruminants Virus in Kenya in 2011

“…The N protein also contains 8 of 9 putative phosphorylation sites recently identified by Sugai et al. () in the measles virus (MV).…”

Organization, Function, and Therapeutic Targeting of the Morbillivirus RNA-Dependent RNA Polymerase Complex

“…This conformational change in MeV N is consistent with the information available for RNA-bound and -free N proteins of HMPV, a member of the Pneumoviridae closely related to the Paramyxoviridae [ 103 ], other paramyxoviruses such as parainfluenza virus 5 and Nipah virus, and distantly related members of the mononegavirales such as VSV and Ebola virus [ 104 , 105 , 106 , 107 ]. Interestingly, the end of the first CTD alpha helix of MeV contains a candidate phosphorylation site (residue T279) that is reportedly involved in regulating transcriptional activity and assembly of the helical RNPs [ 108 ]. Conceivably, the phosphorylation status of N residue 279 could affect the conformation of the CTD and modulate nucleocapsid conformation and accessibility of the RNA to the RdRp.…”

The Nucleocapsid of Paramyxoviruses: Structure and Function of an Encapsidated Template

“…Finally, viral transcription and replication are also affected by the phosphorylation status of N and P. Indeed, both N tail [83][84][85] and the disordered region upstream of P OD [86][87][88][89][90][91][92][93][94] contain major phosphorylation sites targeted by cellular kinases [95][96][97][98][99][100][101]. Although the roles of these phosphorylations are still unclear, data suggest the phosphorylation of some residues can downregulate [85,92,93,100,102] or upregulate RNA synthesis [83,84,91], modify the balance between transcription and replication [103], alter the stability of encapsidated genomes [104], participate in RNA encapsidation [105], tune the interaction between N and P [94], or facilitate the growth of the inclusion bodies [106].…”