Localization of P protein binding sites on the Sendai virus nucleocapsid

Kw, Ryan; Kg, Murti; Portner, Allen

doi:10.1099/0022-1317-71-4-997

Cited by 15 publications

(6 citation statements)

References 6 publications

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“…A deletion of 30 amino acids at the C terminus of Sendai virus P abolishes nucleocapsid binding (40), in accord with the results for measles virus and mumps virus. However, in a cosedimentation assay, the last 95 amino acids of Sendai virus P, encompassing the NBD, did not associate with nucleocapsids under conditions where the full-length P protein was able to bind (42). On the basis of deletion analysis, it was concluded that two noncontiguous regions of Sendai virus P were required for nucleocapsid binding (41,42), the C-terminal 90 amino acids (479 to 568), and an upstream sequence (345 to 412) which is now known to be part of the coiled-coil domain (50).…”

Section: Discussionmentioning

confidence: 99%

“…However, in a cosedimentation assay, the last 95 amino acids of Sendai virus P, encompassing the NBD, did not associate with nucleocapsids under conditions where the full-length P protein was able to bind (42). On the basis of deletion analysis, it was concluded that two noncontiguous regions of Sendai virus P were required for nucleocapsid binding (41,42), the C-terminal 90 amino acids (479 to 568), and an upstream sequence (345 to 412) which is now known to be part of the coiled-coil domain (50). It is possible to reconcile the experimental results for measles virus, mumps virus, and Sendai virus through the following model (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

Kingston

Baase

2004

J Virol

102

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We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system. During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation. For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell. For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses. For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected. These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses. Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD). Spectroscopic studies show that the NBD is predominantly ␣-helical in both viruses. However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P. With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry. The dissociation constant (K d ) was determined to be 13 M at 20°C and 35 M at 37°C. Our data are consistent with a model in which an ␣-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

Kingston

Baase

2004

J Virol

102

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“…For Sendai virus, the amino-terminal half of P has been shown to contain not only the N binding domain (aa 33–41) [52] but also a region from aa 1–144 that is required for synthesis of both mRNA and vRNA [52]. Sendai virus P binds to the N-RNA complex via a region in the C-terminal domain of P during RNA synthesis and vRdRp scanning [53]. In generating rHPIV1-P/C Δ84–85 , we deleted codons 84 and 85 in the C ORF, which corresponded to a complete deletion of codon 88 and partial deletions of codons 87 and 89 in the P ORF (nt: GAG AGT GGA → GGA; aa: ESG → G; Table 1).…”

Section: Discussionmentioning

confidence: 99%

A novel human parainfluenza virus type 1 (HPIV1) with separated P and C genes is useful for generating C gene mutants for evaluation as live-attenuated virus vaccine candidates

Bartlett

Cruz

Boonyaratanakornkit

et al. 2010

Vaccine

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A novel recombinant human parainfluenza virus type 1 (rHPIV1), rHPIV1-C+P, in which the overlapping open reading frames of the C and P genes were separated in order to introduce mutations into the C gene without affecting P, was generated. Infectious rHPIV1-C+P was readily recovered and replicated as efficiently as HPIV1 wild-type (wt) in vitro and in African green monkeys (AGMs). rHPIV1-C+P expressed increased levels of C protein and, surprisingly, activated the type I IFN and apoptosis responses more strongly than HPIV1 wt. rHPIV1-C+P provided a useful backbone for recovering an attenuated P/C gene mutation, (Δ84-85), which was previously unrecoverable, likely due to detrimental effects of the deletion on the P protein. rHPIV1-C Δ84-85 +P and an additional mutant, rHPIV1-C Δ169-170 +P, were found to replicate to similar titers in vitro and to activate the type I IFN and apoptosis responses to a similar degree as rHPIV1-C+P. rHPIV1-C Δ84-85 +P was found to be highly attenuated in AGMs, and all viruses were immunogenic and effective in protecting AGMs against challenge with HPIV1 wt. rHPIV1-C Δ84-85 +P will be investigated as a potential liveattenuated vaccine candidate for HPIV1.

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“…P 4 is proposed to interact with N:RNA in two ways; indirectly via L which interacts with N-core and the template RNA, and directly via the X domains which binds N-tail. It is this latter interaction that tethers P 4 to (mostly inactive) N:RNAs isolated from virions and infected cells (Ryan et al, 1990). Two N ass subunits from an imaginary N:RNA are positioned below the L binding site.…”

Section: Nnv Genome Organization and Expressionmentioning

confidence: 99%

Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

et al. 2004

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mRNA synthesis from nonsegmented negative-strand RNA virus (NNV) genomes is unique in tht the genome RNA is embedded in an N protein assembly (the nucleocapsid) and the viral RNA polymerase does not dissociate from the template after release of each mRNA, but rather scans the genome RNA for the next gene-start site. A revised model for NNV RNA synthesis is presented, in which RNA polymerase scanning plays a prominent role. Polymerase scanning of the template is known to occur as the viral transcriptase negotiates gene junctions without falling off the template.

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Localization of P protein binding sites on the Sendai virus nucleocapsid

Cited by 15 publications

References 6 publications

Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

Characterization of Nucleocapsid Binding by the Measles Virus and Mumps Virus Phosphoproteins

A novel human parainfluenza virus type 1 (HPIV1) with separated P and C genes is useful for generating C gene mutants for evaluation as live-attenuated virus vaccine candidates

Viral RNA polymerase scanning and the gymnastics of Sendai virus RNA synthesis

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