Encapsidation of Sendai virus genome RNAs by purified NP protein during in vitro replication.

The ability of the Sendai virus major nucleocapsid protein, NP, to support the in vitro synthesis and encapsidation of viral genome RNA during Sendai virus RNA replication was studied. NP protein was purified from viral nucleocapsids isolated from Sendai virus-infected BHK cells and shown to be a soluble monomer under the reaction conditions used for RNA synthesis. The purified NP protein alone was necessary and sufficient for in vitro genome RNA synthesis and encapsidation from preinitiated intracellular Sendai virus defective interfering particle (DI-H) nucleocapsid templates. The amount of DI-H RNA replication increased linearly with the addition of increasing amounts of NP protein. With purified detergent-disrupted DI-H virions as the template, however, there was no genome RNA synthesis in either the absence or presence of the NP protein. Furthermore, addition of the soluble protein fraction of uninfected cells alone or in the presence of purified NP protein also did not support DI-H genome RNA synthesis from purified DI-H. Another viral component in addition to the NP protein appears to be required for the initiation of encapsidation, since the soluble protein fraction of infected but not uninfected cells did support DI-H genome replication from purified DI-H.     

Functional mapping of the nucleoprotein of Ebola virus

At 739 amino acids, the nucleoprotein (NP) of Ebola virus is the largest nucleoprotein of the nonsegmented negative-stranded RNA viruses, and like the NPs of other viruses, it plays a central role in virus replication. Huang et al. (Y. Huang, L. Xu, Y. Sun, and G. J. Nabel, Mol. Cell 10:307-316, 2002) 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.     

The conserved N-terminal region of Sendai virus nucleocapsid protein NP is required for nucleocapsid assembly.

Sendai virus nucleocapsid protein NP synthesized in the absence of other viral components assembled into nucleocapsid-like particles. They were identical in density and morphology to authentic nucleocapsids but were smaller in size. The reduction in size was probably due to the fact that they contained RNA only 0.5 to 2 kb in length. Nucleocapsid assembly requires NP-NP and NP-RNA interactions. To identify domains on NP protein involved in nucleocapsid formation, 29 NP protein mutants were tested for the ability to assemble. Any deletion between amino acid residues 1 and 399 abolished formation of nucleocapsid-like particles, but mutants within this region exhibited two different phenotypes. Deletions between positions 83 and 384 completely abolished all interactions. Deletions between residues 1 and 82 and between residues 385 and 399, at the N- and C-terminal ends of the region from 1 to 399, resulted in unstructured aggregates of NP protein, indicating only a partial loss of function. Deletions within the C-terminal 124 amino acids were the only ones that did not affect assembly. The results suggest that NP protein can be divided into at least two separate domains which function independently of each other. Domain I (residues 1 to 399) seems to contain all of the structural information necessary for assembly, while domain II (residues 400 to 524) is not involved in nucleocapsid formation.     

Interaction of chandipura virus N and P proteins: identification of two mutually exclusive domains of N involved in interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N(0)) form (N(0)-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1-180 form the N(0)-P interacting region, whereas, C-terminal residues spanning aa 320-390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N(0).     

The hypervariable C-terminal tail of the Sendai paramyxovirus nucleocapsid protein is required for template function but not for RNA encapsidation.

The paramyxovirus nucleocapsid proteins (NPs) are relatively well conserved, except for the C-terminal 20% (or ca. 100 amino acids), referred to as the tail. We have examined whether this hypervariable tail is required for genome synthesis, both in vitro, where synthesis is predominantly from the input templates, and in vivo, where multiple rounds of amplification occur. In these viruses, genome synthesis and assembly of the nascent chain are coupled. We find that the tail is required in vivo but not in vitro. Closer examination of the in vivo system showed that the tailless NP could encapsidate the genome chain but that amplification did not occur. We interpret these results as indicating that the tail is not required for RNA assembly but is required for the template to function in RNA synthesis. Relatively small deletions within the conserved N-terminal 80% of the protein, on the other hand, rendered the protein nonfunctional in either system. The possible functions of the tail in RNA synthesis are discussed.     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.     

Complexes of Sendai virus NP-P and P-L proteins are required for defective interfering particle genome replication in vitro.

We present evidence that the formation of NP-P and P-L protein complexes is essential for replication of the genome of Sendai defective interfering (DI-H) virus in vitro, using extracts of cells expressing these viral proteins from plasmids. Optimal replication of DI-H nucleocapsid RNA required extracts of cells transfected with critical amounts and ratios of each of the plasmids and was three- to fivefold better than replication with a control extract prepared from a natural virus infection. Extracts in which NP and P proteins were coexpressed supported replication of the genome of purified DI-H virus which contained endogenous polymerase proteins, but extracts in which NP and P were expressed separately and then mixed were inactive. Similarly, the P and L proteins must be coexpressed for biological activity. The replication data thus suggest that two protein complexes, NP-P and P-L, are required for nucleocapsid RNA replication and that these complexes must form during or soon after synthesis of the proteins. Biochemical evidence in support of the formation of each complex includes coimmunoprecipitation of both proteins of each complex with an antibody specific for one component and cosedimentation of the subunits of each complex. We propose that the P-L complex serves as the RNA polymerase and NP-P is required for encapsidation of newly synthesized RNA.     

Dissection of individual functions of the Sendai virus phosphoprotein in transcription.

The Sendai virus P protein is an essential component of the viral RNA polymerase (P-L complex) required for RNA synthesis. To identify amino acids important for P-L binding, site-directed mutagenesis of the P gene changed 17 charged amino acids, singly or in groups, and two serines to alanine within the L binding domain from amino acids 408 to 479. Each of the 10 mutants was wild type for P-L and P-P protein interactions and for binding of the P-L complex to the nucleocapsid template, yet six showed a significant inhibition of in vitro mRNA and leader RNA synthesis. To determine if binding was instead hydrophobic in nature, five conserved hydrophobic amino acids in this region were also mutated. Each of these P mutants also retained the ability to bind to L, to itself, and to the template, but two gave a severe decrease in mRNA and leader RNA synthesis. Since all of the mutants still bound L, the data suggest that L binding occurs on a surface of P with a complex tertiary structure. Wild-type biological activity could be restored for defective polymerase complexes containing two P mutants by the addition of wild-type P protein alone, while the activity of two others could not be rescued. Gradient sedimentation analyses showed that rescue was not due to exchange of the wild-type and mutant P proteins within the P-L complex. Mutants which gave a defective RNA synthesis phenotype and could not be rescued by P establish an as-yet-unknown role for P within the polymerase complex, while the mutants which could be rescued define regions required for a P protein function independent of polymerase function.     

Interaction between Hantavirus Nucleocapsid Protein (N) and RNA-Dependent RNA Polymerase (RdRp) Mutants Reveals the Requirement of an N-RdRp Interaction for Viral RNA Synthesis

Viral ribonucleocapsids harboring the viral genomic RNA are used as the template for viral mRNA synthesis and replication of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Here we show that hantavirus nucleocapsid protein (N protein) interacts with RdRp in virus-infected cells. We mapped the RdRp binding domain at the N terminus of N protein. Similarly, the N protein binding pocket is located at the C terminus of RdRp. We demonstrate that an N protein-RdRp interaction is required for RdRp function during the course of virus infection in the host cell.     

In vitro assembly of Ebola virus nucleocapsidlike complex expressed in E. coli

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryoeletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451–739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451–739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.     

The C-terminal LCAR of host ANP32 proteins interacts with the influenza A virus nucleoprotein to promote the replication of the viral RNA genome

The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs.