Newcastle disease virus nucleocapsid protein: self-assembly and length-determination domains

Kho, Chiew Ling; Tan, Wen Siang; Tey, Beng Ti; Yusoff, Khatijah

doi:10.1099/vir.0.19107-0

Cited by 34 publications

(27 citation statements)

References 22 publications

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“…Constructs in which most or all of the tail was removed (1 to 375, 1 to 400, and 1 to 425) produced the longest particles. Very similar observations were recently reported for the Newcastle disease virus N protein (28).…”

Section: Discussionsupporting

confidence: 75%

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: Discussionsupporting

confidence: 75%

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

Kingston

Baase

2004

J Virol

102

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“…3 to 5). Several studies have demonstrated that the N-terminal regions of the NP/N molecules of paramyxoviruses are generally important for NP-NP (N-N) interaction (1,4,19,20,27,29), therefore our data support the functional conservation of this region of NP for NP-NP interactions among the members of Mononegavirales. The fact that a small region (residues 230 to 370) of the N terminus of Ebola virus NP shares appreciable amino acid homology with the NPs/Ns of paramyxoviruses and, to a lesser extent, with those of rhabdoviruses (34) further supports this notion.…”

Section: Discussionsupporting

confidence: 67%

Functional Mapping of the Nucleoprotein of Ebola Virus

Watanabe

Noda

Kawaoka

2006

J Virol

155

177

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) previously demonstrated that NP, together with the minor matrix protein VP24 and polymerase cofactor VP35, is necessary and sufficient for the formation of nucleocapsid-like structures that are morphologically indistinguishable from those seen in Ebola virus-infected cells. They further showed that NP is O glycosylated and sialylated and that these modifications are important for interaction between NP and VP35. However, little is known about the structure-function relationship of Ebola virus NP. Here, we examined the glycosylation of Ebola virus NP and further investigated its properties by generating deletion mutants to define the region(s) involved in NP-NP interaction (self-assembly), in the formation of nucleocapsid-like structures, and in the replication of the viral genome. We were unable to identify the types of glycosylation and sialylation, although we did confirm that Ebola virus NP was glycosylated. We also determined that the region from amino acids 1 to 450 is important for NP-NP interaction (self-assembly). We further demonstrated that these amino-terminal 450 residues and the following 150 residues are required for the formation of nucleocapsid-like structures and for viral genome replication. These data advance our understanding of the functional region(s) of Ebola virus NP, which in turn should improve our knowledge of the Ebola virus life cycle and its extreme pathogenicity.Ebola and Marburg viruses are filamentous, enveloped, nonsegmented, negative-stranded RNA viruses of the family Filoviridae in the order Mononegavirales, which also includes the Paramyxoviridae, Rhabdoviridae, and Bornaviridae (7, 32). Despite the fact that the severe hemorrhagic fever caused by Ebola virus is associated with extremely high mortality rates in human and nonhuman primates, an effective vaccine or antiviral drugs have yet to be developed.Ebola virus particles consist of at least seven structural proteins encoded by a single-stranded negative-sense RNA genome. Four of these proteins-nucleoprotein (NP), VP35, VP30, and the RNA-dependent RNA polymerase (L)-are components of the ribonucleoprotein complex that is responsible for the transcription and replication of the viral genome (26). Three others-glycoprotein (GP), VP40, and VP24-are membrane-associated proteins (12, 32), and VP24 is also thought to be involved in nucleocapsid formation (15).The NP of Ebola virus is the largest (739 amino acid residues) nucleoprotein of the nonsegmented negative-stranded RNA viruses and can be divided into a hydrophobic N-terminal half (approximately 350 amino acids) and a hydrophilic C-terminal half (33). Huang et al. (15) showed that Ebola virus NP is O glycosylated and sialylated and that these modifications are required for its interaction with VP35, which suggests that the modifications are important for viral genome replication. However, the functional region(s) of Ebola virus NP has not been characterized. The nucleoproteins (NP/N) of nonsegmented negative-stranded RNA viruses, including Marburg virus NP, are known to...

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“…2c), demonstrating that the R2 and R3 regions are interchangeable with those from NDV and the differences in the amino acid residues in these regions (Fig. 3) are unlikely to be involved in NiV capsid formation.Many studies have shown that paramyxovirus nucleocapsid proteins self-assemble into herringbone-like particles when expressed in a heterologous system in the absence of the viral proteins (Buchholz et al, 1993; Curran et al, 1993; Bankamp et al, 1996;Krishnamurthy & Samal, 1998;Seal et al, 2002;Kho et al, 2003). However, the contiguous regions of the N protein required for capsid assembly are poorly characterized in Henipavirus.…”

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confidence: 99%

Mutagenesis of the nucleocapsid protein of Nipah virus involved in capsid assembly

Ong

Yusoff

Kho

et al. 2009

Journal of General Virology

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The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino-and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30-32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.Nipah virus (NiV), the second member of the genus Henipavirus (Wang et al., 2000(Wang et al., , 2001, has been associated with several outbreaks of viral encephalitis in humans in South-east Asia. The first and largest NiV outbreak in Malaysia ended a year after its identification in 1998. During this period, more than 100 human lives were claimed and the pig industry in the country was paralysed. Pig farms were forced to close and millions of pigs were culled in order to control the viral outbreak (Chua et al., 1999(Chua et al., , 2000. Two years later, an outbreak in Bangladesh alerted the world to the reoccurrence of this virus (Hsu et al., 2004;Harcourt et al., 2005). Shocking news were reported in 2004 from India where the mortality rate of the infected patients was more than 70 % (Diederich & Maisner, 2007). The spread of the virus from human to human was suspected, and no pig intermediary was reported (Chadha et al., 2006). Several species of flying foxes, in the genus Pteropus, have been identified as the natural reservoirs of NiV (Eaton et al., 2006).NiV has a single-stranded negative-sense RNA of approximately 18.2 kb which encodes six major structural proteins: nucleocapsid (N), phospho-(P), matrix (M), fusion (F), glyco-(G) and large (L) proteins (Wang et al., 2001). The most abundant structural protein, N, and the genomic RNA constitute the core helical nucleocapsid structure of NiV. The genomic RNA is associated with the N, P and L proteins to form the ribonucleoprotein complex (Diederich & Maisner, 2007).Structural and functional studies of the N protein of paramyxoviruses have been the main focus for years, due to its crucial functional role during the replication of the genomic RNA. Transmission electron microscopic studies revealed that all the paramyxovirus nucleocapsids are helical in structure, but there are significant differences among the virus genera (Bhella et al., 2002) such as Sendai virus (SeV; Myers et al., 1999), measles virus (MeV;Bhella et al., 2004) and respiratory syncytial virus (RSV; MacLellan et al., 2007). This is most likely due to the differences in the primary structure of the N proteins of these viruses which determine the tertiary and quaternary structures of the nucleocapsids.For members of the genus H...

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Newcastle disease virus nucleocapsid protein: self-assembly and length-determination domains

Cited by 34 publications

References 22 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

Functional Mapping of the Nucleoprotein of Ebola Virus

Mutagenesis of the nucleocapsid protein of Nipah virus involved in capsid assembly

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