Sendai Virus Nucleocapsid Protein Research Articles

Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp)

We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

Generation of Sendai virus nucleocapsid-like particles in yeast

The gene encoding Sendai virus nucleocapsid protein was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. The high level of recombinant Sendai virus nucleocapsid protein expression (12–14 mg/l of yeast culture) was obtained. The evaluation of recombinant proteins expression in yeast by Western blot analysis revealed specific reactivity with immune sera. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid protein. These structures contained host RNA, which was resistant to an RNase treatment. The nucleocapsid protein revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. The development of a simple, efficient and cost-effective system for generation of Sendai virus nucleocapsid protein might help to upgrade reagents for virus serology, and facilitate investigation of virus replication and RNA encapsidation mechanisms.

A Highly Conserved Region of the Sendai Virus Nucleocapsid Protein Contributes to the NP–NP Binding Domain

The nucleocapsid protein (NP) of Sendai virus is an essential component of both the nucleocapsid template and the NP–NP and NP0–P protein complexes required for viral RNA replication. When expressed alone in mammalian cells NP self-assembles into nucleocapsidlike particles which appear to contain cellular RNA. To identify putative NP–NP binding domains, fusions between the monomeric maltose-binding protein (MBP) and portions of NP were constructed. The fusion proteins which contain the central conserved region (CCR) (amino acids 258–357, MBP–NP1) and the N-terminal 255 amino acids (MBP-NP2) of NP both oligomerized, suggesting that these regions contain sequences important for NP–NP self-assembly. In addition, the MBP–NP1 fusion protein can function as an inhibitor of viral RNA replication. Complementary studies involving site-directed mutagenesis of the full-length NP protein have identified specific residues in the CCR which are essential for viral RNA replicationin vitro.Two such replication-negative mutants, F324V and F324I, were defective in self-assembly, suggesting that the Phe residue at amino acid 324 is essential for the NP–NP interaction. A third mutant, NP260-1 (Y260D), self-assembled to form aberrant oligomers which exhibit an unusual helical structure and appear to lack any associated RNA. The mutants NP299-5 (L299I and I300V) and NP313-2 (I313F), in contrast, appear to form all the required protein complexes, but were inactive in viral RNA replication, suggesting that interactions specifically with Sendai RNA were disrupted. These data have thus identified specific residues in the CCR of the native NP protein which appear to be important for NP–NP or NP–RNA interactions and for genome replication.

An amino-terminal domain of the Sendai virus nucleocapsid protein is required for template function in viral RNA synthesis.

The nucleocapsid protein (NP) of Sendai virus encapsidates the genome RNA, forming a helical nucleocapsid which is the template for RNA synthesis by the viral RNA polymerase. The NP protein is thought to have both structural and functional roles, since it is an essential component of the NP0-P (P, phosphoprotein), NP-NP, nucleocapsid-polymerase, and RNA-NP complexes required during viral RNA replication. To identify domains in the NP protein, mutants were constructed by using clustered charge-to-alanine mutagenesis in a highly charged region from amino acids 107 to 129. Each of the mutants supported RNA encapsidation in vitro. The product nucleocapsids formed with three mutants, NP114, NP121, and NP126, however, did not serve as templates for further amplification in vivo, while NP107, NP108, and NP111 were nearly like wild-type NP in vivo. This template defect in the NP mutants from amino acids 114 to 129 was not due to a lack of NP0-P, NP-NP, or nucleocapsid-polymerase complex formation, since these interactions were normal in these mutants. We propose that amino acids 114 to 129 of the NP protein are required for the nucleocapsid to function as a template in viral genome replication.

Expression of Sendai virus nucleocapsid protein in a baculovirus expression system and application to diagnostic assays for Sendai virus infection.

The most common diagnostic technique for the detection of Sendai virus infection in rodents is serological evaluation by enzyme-linked immunosorbent assay (ELISA) with semipurified preparations of whole virions as antigens. This assay often suffers from a lack of specificity. The goal of the present project was to develop more specific antigens for use in diagnostic testing by producing recombinant antigens in insect cells. To identify viral proteins immunoreactive in multiple laboratory rodent species, Western blots (immunoblots) of viral polypeptides were probed with immune sera from mice, rats, and hamsters. The nucleocapsid protein (NP) reacted with immune sera from all species tested. Therefore, the NP gene was selected for cloning and expression in a baculovirus. To construct the recombinant, complementary DNA was synthesized by reverse transcription PCR from Sendai virus RNA with primers from the 5' and 3' termini of the NP-coding region. Amplified DNA was cloned into a baculovirus transfer vector (pBlueBacHis A) and was cotransfected with wild-type baculovirus into insect cells. Baculovirus recombinants containing the NP gene were identified by PCR. Evaluation of the recombinant proteins expressed in insect cells by Western blot analysis revealed specific reactivity with immune sera. In comparison with conventional ELISAs that use whole virions as the antigen, ELISAs that use recombinant NP were more specific.

The Carboxy-terminal Domain of Sendai Virus Nucleocapsid Protein Is Involved in Complex Formation between Phosphoprotein and Nucleocapsid-like Particles

The replicative unit of Sendai virus, the nucleocapsid, is composed of the viral genomic RNA and the viral proteins NP, P, and L. P and L proteins form a polymerase complex and are associated with the nucleocapsid core (NP/RNA complex). The NP protein has recently been shown to be composed of two domains. While the amino-terminal domain I encapsidates RNA and gives rise to the characteristic helical nucleocapsid structure, the precise function of the carboxy-terminal domain II remained unclear. It was however supposed to be involved in binding the polymerase complex. To test this hypothesis, we established a cosedimentation assay which detects complex formation between P protein and nucleocapsid-like (NC-like) particles. Four mutant NC-like particles all carrying amino acid deletions in domain II of the NP protein were tested. Complex formation was abolished after deletion of amino acids 426-497 or 456-524, while deletions of amino acids 400-415 or 414-439 had no influence on the interaction. The results indicate that domain II of NP protein is involved in binding P protein to nucleocapsids. The function and position of a putative P protein binding site in domain II are discussed.

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.

Mapping of antigenic domains of Sendai virus nucleocapsid protein expressed in Escherichia coli

Several nonoverlapping epitopes were mapped on the primary sequence of the Sendai virus NP protein. After a complete cDNA clone of the Sendai virus NP gene was expressed in Escherichia coli, deletion constructs were used to generate a series of overlapping NP fragments deleted at their C termini. Immunoblot analyses with 11 monoclonal antibodies identified four antigenic sites. All of these sites resided in the C-terminal half of NP and were also the only sites detected with a polyclonal serum. These findings confirm and extend the evidence that the C terminus of the NP protein represents the domain exposed on the surface of the nucleocapsid. One of the monoclonal antibodies reacted with a site, comprising only 6 amino acids, lying with a hinge between an alpha-helix and a beta-strand in the predicted secondary structure of NP. Since this antibody is a potent inhibitor of in vitro viral RNA synthesis (K. L. Deshpande and A. Portner, Virology 139:32-42, 1984), the epitope may be critical to the flexibility of the NP molecule that makes the RNA template accessible during RNA synthesis.

Antisense oligodeoxynucleotides provide insight into mechanism of translation initiation of two Sendai virus mRNAs.

Translation of Sendai virus nucleocapsid protein (NP) and phosphoprotein (P/C) mRNAs in rabbit reticulocyte lysates in the presence of antisense oligodeoxynucleotides (15-20-mers) showed that oligonucleotides having complementarity within the 5' noncoding region of the mRNAs blocked translation, oligonucleotides having complementarity within 20 nucleotides upstream from the initiator codon blocked translation only partially, and oligonucleotides complementary to the coding region of mRNA had no effect on translation. The results suggest the possibility that the 80 S initiation complex may form about 20 bases upstream from the initiator codon. Alternatively, the 40 S preinitiation complex may recognize an initiator codon at least 20 nucleotides upstream from the codon, activating a helix-destabilizing process.

Conserved structures among the nucleocapsid proteins of the paramyxoviridae: Complete nucleotide sequence of human parainfluenza virus type 3 NP mRNA

The nucleotide sequence of the mRNA coding for the nucleocapsid protein (NP) of the paramyxovirus, human parainfluenza virus type 3 (PIV-3), has been determined. The NP mRNA was found to contain 1642 bases, excluding poly(A), and encode a protein of 515 amino acids, with a molecular weight of 57,823. Amino acid residues 1 through 420 of PIV-3 NP protein showed extensive sequence homology with the corresponding amino acids of Sendai virus nucleocapsid protein. There was virtually no homology between the last 95 amino acids. Comparison of the NP proteins of PIV-3, Sendai virus, measles virus, and canine distemper virus revealed, from amino acid residues 160 through 390, some conserved areas between the corresponding proteins of these paramyxoviruses. The 5′ terminal sequence of PIV-3 NP mRNA (5′-AGGATTAAAG-3′) was similar to the conserved sequence 5′-AGGG T ANAA A TG-3′ found at the 5′ termini of Sendai virus mRNAs. Both PIV-3 NP and Sendai virus mRNAs had a common 3′ terminal tetranucleotide (5′-TAAG-3′) preceding the poly (A) tail.

Molecular cloning and sequence analysis of the human parainfluenza 3 virus RNA encoding the nucleocapsid protein

The sequence of 1690 nucleotides from the 5′ end of the viral complementary RNA for the human parainfluenza 3 virus was determined by molecular cloning. One large open reading frame consisting of 1548 nucleotides was demonstrated. The encoded protein, the nucleocapsid protein (NP), consists of 515 amino acids, and has a predicted molecular weight of 57,819. A noncoding 5′ sequence of 51 nucleotides is present at the end of the NP-mRNA. Two consensus sequences were identified which are homologous with sequences found in Sendai virus. One of these sequences, AGGATTAAAG, was located at the 5′ end of the nucleocapsid mRNA and may function in transcription initiation. The other consensus sequence, GTAAGGGAA, was found in the viral genomic leader sequence. The nucleocapsid protein amino acid sequence was compared to other members of the Paramyxoviridae family. The parainfluenza 3 virus protein nucleocapsid amino acid sequence demonstrated a high degree of homology with the Sendai virus nucleocapsid protein. Seventy percent of the first 387 amino acids from the amino termini were identical. Little homology was observed in the distal carboxy termini.

Monoclonal antibodies to the P protein of sendai virus define its structure and role in transcription

Four monoclonal antibodies specific for Sendai virus nucleocapsid protein P were used to examine both the antigenic structure of P and its role in transcription. Three distinct antigenic regions were delineated on P by competitive radioimmunoassays (RIAs), and through Western blot analysis all three sites were mapped to a 40,000-MW (40K) Staphylococcus aureus protease V8-digestion fragment, which remains associated with the neucleocapsid structure. To study the function of P, nucleocapsids were treated with saturating amounts of anti-P monoclonal antibodies and it was found that transcription in vitro was inhibited by 60–90%. Data, therefore, are consistent with the conclusion that the P protein is required for transcription and that the 40K protease-resistant core contains the functionally important portion of the molecule. Further analysis of the P structure showed that some of the 40K fragments were linked by disulfide bonds. These results suggest that the protease-resistant 40K fragment is in the carboxylterminal half of P, since the three cysteine residues of P are found there ( C. Giorgi, B. M. Blumberg, and D. Kolakofsky (1983), Cell 35, 829–836).

Structural and functional analysis of sendai virus nucleocapsid protein NP with monoclonal antibodies

Monoclonal antibodies specific for Sendai virus nucleocapsid protein NP were used to map the antigenic structure of NP and to investigate the role of NP in transcription. Using nine anti-NP antibodies in competitive-binding (CB) assays, it was found that the NP molecule contained at least two topographically distinct antigenic sites. By Western blot analysis, one of the NP epitopes belonging to antigenic site I was localized to a M r 34,000 (34K) trypsin digest fragment, and another to a M r 48,000 (48K) fragment which remained associated with the nucleocapsid. The other antibodies which define antigenic site I did not react with either fragment; however, the results of CB would indicate that their epitopes were in a region on the tertiary structure of the NP molecule that is closely proximal to these fragments. The 48K and 34K fragments on the published NP amino acid sequence have been tentatively identified. Since the 34K and 48K fragments bind antibody, it appears that nucleocapsid-bound NP may be folded into a configuration which places at least some of these sequences on the surface of the nucleocapsid structure. Six antibodies representing both antigenic sites were purified for functional studies. All the antibodies inhibited nucleocapsid transcription in vitro to the same extent (>90%); however, they differed in the amount of antibody required to produce the same effect. Within site I, antibodies producing maximum inhibition were divided into three groups: three antibodies inhibited at relatively low concentrations (0.17 μg), one antibody inhibited at an intermediate range (0.43 μg), and another required a 10-fold higher concentration (1.73 μg) to produce the same effect. The antibody which detected the 48K trypsin digest fragment was the one which fell into the intermediate range for transcription inhibition, while the antibody that detected the 34K fragment was in the low range. Thus, antigenic site I, defined by CB and trypsin digestion studies, can be defined further into three subsites which appear to differ in their involvement in the transcription process. Antigenic site II was defined by a single antibody which also inhibited transcription by >90%.

The relationship of conformational changes in the Sendai virus nucleocapsid to proteolytic cleavage of the NP polypeptide.

Abstract Previous studies showed that the conformation of the paramyxovirus nucleocapsid varies extensively with regard to the flexibility and coiling of the helix, and changes of this type could be important for virus assembly and transcription of the viral RNA. The helix is tightly coiled and relatively inflexible at high salt concentration (e.g., 1.0 M NaCl) and loosely coiled or extended in low-salt buffer (0.01 M). It has now been found that a salt-induced conformational change can alter the accessibility of the Sendai virus nucleocapsid protein (NP, Mr ∼ 60,000) to cleavage by trypsin. When the nucleocapsid is tightly tightly coiled, NP is cleaved to yield a single major cleavage product (t48, Mr ∼ 48,000) that contains the original N-terminus of the molecule. When the nucleocapsid is in the uncoiled state, t48 is further cleaved to a major N-terminal product, t34, and a second product t15; t15 may be further cleaved to yield t12. Unlike the ∼12,000 daltons of NP that is degraded to small peptides and lost from the nucleocapsid when NP is cleaved to t48, both t34 and t15 (or t12) remain associated with the nucleocapsid structure although thoy are not disulfide bonded to each other. Sendai virus nucleocapsids containing t34 and t15 (or t12) have the same morphological appearance and respond reversibly to changes in salt concentration as do nucleocapsids composed of the native NP or t48. The results indicate that the properties of the NP subunit that are important in determining the helical conformation of the nucleocapsid reside in the N-terminal 48,000 daltons of the molecule and are unaffected by the further cleavages. In contrast to the conformation-dependent exposure of tryptic cleavage sites on NP, the viral RNA in the nucleocapsid is inaccessible to digestion by ribonuclease regardless of salt-dependent changes in conformation, or of the state of tryptic cleavage of NP. Thus neither the tightness of stacking of the helix nor proleolytic cleavages in the NP protein affect the binding of the RNA to NP or its protection from ribonuclease.

Specific cleavage of Sendai virus nucleocapsid protein subunits during virus storage

The alteration of whole Sendai virus and especially of its nucleocapsid polypeptides, during storage of the virus at 4 degree C in the allantoic fluids in which it was cultivated, has cultivated, has been studied by sodium dodecyl sulfate gel electrophoresis. During virus storage the nucleocapsid protein subunits with a molecular weight of 60,000 and the putative inner envelope protein with a molecular weight of 38,000 were mainly affected. Both virus components were partially degraded to smaller components. Examination of nucleocapsids isolated from "stored" virus showed that, in addition to the 60,000-molecular weight polypetide component, a smaller polypeptide component with a molecular weight of 46,000 appeared. The relative proportion of the small component increased with the storage period: a kind of specific conversion of large to small components occurred during storage. Since viruses kept in the absence of allantoic fluids revealed no similar modifications of their polypeptides, we concluded that a cellular component present in the allantoic fluids - very likely of enzymatic nature - is responsible for the observed cleavage of virus polypeptides.