Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein

155

177

) 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...

Section: Discussionsupporting

confidence: 84%

Functional Mapping of the Nucleoprotein of Ebola Virus

Watanabe

Noda

Kawaoka

2006

155

177

“…It is tempting to speculate that Le nt 16 to 34 encode a sequence in the complementary nascent antigenome that binds N and acts as an encapsidation nucleation site. However, an attempt to identify an N-binding site in the RSV Le-encoded transcript failed to show evidence for a specific interaction (28). In addition, a previous study showed that LeC-mRNA transcripts were not encapsidated even under conditions in which N protein was in excess (14), suggesting that in RSV, encapsidation might not be initiated simply by interaction of N with a cisacting sequence at the 5Ј end of the RNA.…”

Section: Discussionmentioning

confidence: 98%

Identification of Internal Sequences in the 3′ Leader Region of Human Respiratory Syncytial Virus That Enhance Transcription and Confer Replication Processivity

McGivern

Collins

Fearns

2005

Previous studies of respiratory syncytial virus have shown that the 44-nucleotide (nt) leader (Le) region is sufficient to initiate RNA replication, producing antigenome RNA, and that the Le and adjoining gene start (GS) signal of the first gene are sufficient to initiate transcription, producing mRNA. A cis-acting element necessary for both transcription and replication was mapped within the first 11 nt at the 3 end of Le. In the present study the remainder of the Le region was mapped to identify sequences important for transcription and replication. A series of minigenomes with mutant Le sequences was generated, and their ability to direct transcription and replication was determined by Northern blot analysis, which examined full-length antigenome and mRNA, and by primer extension analysis, which examined antigenome and mRNA initiation. With regard to transcription, nt 36 to 43, located immediately upstream of the GS signal, were found to be necessary for optimal levels of mRNA synthesis, although the GS signal in conjunction with the 3-terminal region of Le was sufficient to direct accurate mRNA synthesis initiation. With regard to replication, the first 15 nt of Le were found to be sufficient to direct initiation of antigenome synthesis, but nt 16 to 34 were required in addition for efficient encapsidation and production of full-length antigenome. Analysis of transcripts produced from di-and tricistronic minigenomes indicated that a significant proportion of abortive replicases continue RNA synthesis to the end of the first gene and then continue in a transcription mode along the remainder of the genome.

“…Although N-RNA rings used for three-dimensional (3D) structure determinations were cocrystallized with P CTD , no electron densities corresponding to the latter were observed, and the P binding site on the N-RNA complex remained to be determined. Several studies sought to address this point but led to conflicting results (13,31,32,43). More specifically, the implication of the N NTD and/or N CTD in the interaction with P remains to be clarified.…”

mentioning

confidence: 99%

Characterization of a Viral Phosphoprotein Binding Site on the Surface of the Respiratory Syncytial Nucleoprotein

Galloux

Tarus

Blazevic

et al. 2012

The human respiratory syncytial virus (HRSV) genome is composed of a negative-sense single-stranded RNA that is tightly associated with the nucleoprotein (N). This ribonucleoprotein (RNP) complex is the template for replication and transcription by the viral RNA-dependent RNA polymerase. RNP recognition by the viral polymerase involves a specific interaction between the C-terminal domain of the phosphoprotein (P) (P CTD ) and N. However, the P binding region on N remains to be identified. In this study, glutathione S-transferase (GST) pulldown assays were used to identify the N-terminal core domain of HRSV N (N NTD ) as a P binding domain. A biochemical characterization of the P CTD and molecular modeling of the N NTD allowed us to define four potential candidate pockets on N (pocket I [PI] to PIV) as hydrophobic sites surrounded by positively charged regions, which could constitute sites complementary to the P CTD interaction domain. The role of selected amino acids in the recognition of the N-RNA complex by P was first screened for by site-directed mutagenesis using a polymerase activity assay, based on an HRSV minigenome containing a luciferase reporter gene. When changed to Ala, most of the residues of PI were found to be critical for viral RNA synthesis, with the R132A mutant having the strongest effect. These mutations also reduced or abolished in vitro and in vivo P-N interactions, as determined by GST pulldown and immunoprecipitation experiments. The pocket formed by these residues is critical for P binding to the N-RNA complex, is specific for pneumovirus N proteins, and is clearly distinct from the P binding sites identified so far for other nonsegmented negative-strand viruses.H uman respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (7). It infects close to 100% of infants within the first 2 years of life and is the main cause of bronchiolitis. Also, bovine respiratory syncytial virus (BRSV), which is very similar to its human counterpart, is a major cause of respiratory disease in calves, resulting in substantial economic losses to the cattle industry worldwide (49). RSV belongs to the genus Pneumovirus of the family Paramyxoviridae and the order Mononegavirales (7). The viral genome consists of a nonsegmented ϳ15-kb RNA of negative polarity which encodes 11 proteins. As for all the members of the Mononegavirales, the genomic RNA of RSV is always tightly bound by the viral nucleoprotein (N) and maintained as a helical N-RNA ribonucleoprotein (RNP) complex (9). The RNP is used as the template for transcription and replication by the RNAdependent RNA polymerase (RdRp) complex, consisting of L (large protein) and its cofactor P (phosphoprotein). Whereas N, P, and L are sufficient to mediate viral replication (15, 50), transcription activity requires L, P, and the M2-1 protein, which functions as a processivity polymerase cofactor (8).The specific recognition of the viral N-RNA matrix by the RdRp constitutes a prerequisite for vir...