Sendai virus nucleocapsids in the nucleoli of infected cells

Summary Uncoiled parental Sendai virus nucleocapsids were revealed in the nucleoli of Sendai virus infected Ehrlich ascites tumor cells early in infection. Their sedimentation coefficient and buoyant density were similar to that of parental nucleocapsids isolated from the cytoplasm (150–180S in a sucrose gradient and 1.35–1.37 g/ml in a CsCl gradient). Later in infection the nucleocapsids were involved into complexes with a sedimentation coefficient about 500S. Similar results were obtained with nucleoli of chicken embryo lung cells. The complex in Ehrlich tumor cells contained 23–26S nascent RNA, its synthesis being inhibited by cycloheximide.     

Mapping the phosphoprotein binding site on Sendai virus NP protein assembled into nucleocapsids

To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.     

Antibodies against Sendai virus L protein: distribution of the protein in nucleocapsids revealed by immunoelectron microscopy

Antibodies against the L protein of Sendai virus were made by immunizing rabbits with a synthetic peptide representing a carboxyl-terminal region of the protein predicted from the base sequence of its gene. These antibodies were used to localize the L protein in viral nucleocapsids by electron microscopy. Immunogold labeling revealed that L protein molecules were distributed in clusters along nucleocapsids, suggesting that L molecules act cooperatively in viral RNA synthesis. Immunogold double-labeling showed that all L clusters were associated with clusters of P molecules. We believe that this morphological association reflects the functional cooperation of the L and P proteins in viral RNA synthesis.     

Separate domains of Sendai virus P protein are required for binding to viral nucleocapsids

The role of Sendai virus P protein in viral RNA synthesis involves association with the nucleocapsid template. There is evidence that the carboxyl-terminal region of P protein is responsible for this association (K. W. Ryan and D. W. Kingsbury, 1988, Virology 167, 106-112). To define the P protein sequences involved more precisely, deletions were generated in a cDNA clone of the P gene. Proteins synthesized in vitro from these altered P genes were mixed with extracts from infected cells to determine if they could attach to nucleocapsids. Under conditions where full-size P protein was able to bind, a protein comprising the 95 carboxyl-terminal residues of P protein (Sendai virus X protein) did not bind. This indicated that other P protein residues were required, in addition to the 95 residues at the carboxyl-terminal end. To locate these other residues, P genes were constructed with overlapping deletions of sequences encoding the carboxyl-terminal 40% of the protein. Analysis of these deleted proteins revealed that the necessary residues were in two separate binding domains, amino acids 345 to 412 and 479 to 568 (the carboxyl-terminus). Deletion of the 66 residues between these regions did not affect attachment. Therefore, the formation of a functional binding site requires residues within two separate regions of P protein.     

Budding efficiency of Sendai virus nucleocapsids: influence of size and ends of the RNA

The budding efficiency of Sendai virus antigenomes, as well as of defective interfering (DI) nucleocapsids of the deletion and copy-back types, was compared to that of the viral genome during infections of baby hamster kidney (BHK) cells. The antigenomes were shown to bud into virus particles as efficiently as the genomes, arguing for the irrelevance of the nucleocapsid-RNA ends in regulating the efficiency of budding. The DI nucleocapsids, however, were restricted in their budding by factors inversely proportional to their size, arguing for an effect of nucleocapsid size in this process. This restriction in budding, however, appeared to be only expressed under conditions of very efficient DI-RNA replication.     

Intracellular phosphorylation of the Sendai virus P protein

Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.     

Paradoxical effects of Sendai virus DI RNA size on survival: inefficient envelopment of small nucleocapsids

In the assembly of nonsegmented negative-stranded RNA viruses, such as Sendai virus, the envelopment process allows extensively deleted genomes to survive by transmission from cell to cell in virus particles. To assess the impact of the sizes of such defective-interfering (DI) genomes on their survival, we performed competition tests among various species. Among copy-back DI RNAs, a 450-base species was gradually eliminated from DI virions by a 1200-base species, and the latter was independently eliminated by a 2800-base species. In each case, the smaller RNA species was synthesized and encapsidated at least as efficiently as the larger species, revealing that the level of competition was at the envelopment step in virus assembly. In contrast to the results obtained with the copy-back DI RNAs, repeated high multiplicity passage of a family of four internally deleted RNAs eliminated all but the smallest species, comprising about 1600 bases. Both sets of findings can be reconciled by the hypothesis that the efficiency of DI nucleocapsid envelopment decreases progressively when the RNA is smaller than about 1600 bases.     

Topological organization of Sindbis virus capsid protein in isolated nucleocapsids

Sindbis virus nucleocapsids were isolated from mature virions by a two-step purification method. Detergent-treated virions were sedimented in sucrose gradients and the nucleocapsid peaks chromatographed on RNase-free Sephadex G-200. The purified nucleocapsids displayed several morphologies when examined in the electron microscope. These morphologies, and the results of double-angle shadowing, suggest that the core of this enveloped virus has the shape of a regular icosahedron with a triangulation number of 4. Peptide mapping of capsid protein obtained from nucleocapsids that had been radioiodinated by a variety of means, indicated that of the four tyrosine residues in the protein, only Tyr180 was exposed at the surface of the icosahedral structure. The other three residues were not exposed on the outer surface of the nucleocapsid shell, nor on the surface of capsid protein itself, implying that they were buried within the folded protein.     

Purification of the Sendai virus nonstructural C protein expressed in E. coli,and preparation of antiserum against C protein

Summary An expression plasmid, ptac-C, was constructed by inserting the cDNA of the coding region of the Sendai virus nonstructural C protein downstream of the tac promoter ofE. coli expression plasmid ptac12-Bam. A new protein produced inE. coli after induction was purified to near homogeneity. The purified protein was found to be identical with the C protein predicted from the C gene cDNA in molecular weight, isoelectric point, amino acid composition, and the amino acid sequence at the N-terminal of the protein as well as those of several fragments obtained on V8 protease digestion. Antiserum raised against the purified protein specifically reacted with the C protein in infected cells. Using this antiserum, the localization of the C protein in infected cells was examined by immunofluorescence, which revealed that it appeared in the cytoplasm but not in nuclei.     

Fuzzy material surrounding measles virus nucleocapsids identified as matrix protein

Summary Measles virus grown in Vero cell cultures was examined at the ultra-structural level after immunoperoxidase staining with antiserum against the matrix protein. The antiserum clearly preferentially labeled the fuzzy material surrounding cytoplasmic nucleocapsids, but not the nucleocapsids themselves.With 4 Figures     

Characterization of the Sendai virus V protein with an anti-peptide antiserum.

The Sendai virus V protein, which is a fusion of the P and V ORFs of the P gene, was characterized with antisera to a portion of the V ORF and compared to the P protein. The only property found in common with P is that V is also highly phosphorylated, and this is so even when these proteins are expressed independently of the other viral proteins. Otherwise, V was not found in virions, was not strongly associated with viral nucleocapsids like P, and anti-V had no effect on viral RNA synthesis in vitro under conditions where anti-P was highly inhibitory. The available evidence suggests that V may play a role in RNA synthesis, but it is not an essential one like that of the P protein.     

Sequence-function analysis of the Sendai virus L protein domain VI

The large (about 2200 amino acids) L polymerase protein of nonsegmented negative-strand RNA viruses (order Mononegavirales) has six conserved sequence regions (“domains”) postulated to constitute the specific enzymatic activities involved in viral mRNA synthesis, 5′-end capping, cap methylation, 3′ polyadenylation, and genomic RNA replication. Previous studies with vesicular stomatitis virus identified amino acid residues within the L protein domain VI required for mRNA cap methylation. In our recent study we analyzed four amino acid residues within domain VI of the Sendai virus L protein and our data indicated that there could be differences in L protein sequence requirements for cap methylation in two different families of Mononegavirales — rhabdoviruses and paramyxoviruses. In this study, we conducted a more comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four L mutants, and testing these mutations for their effects on viral mRNA synthesis, cap methylation, viral genome replication and virus growth kinetics. Our analysis identified several residues required for successful cap methylation and virus replication and clearly showed the importance of the K-D-K-E tetrad and glycine-rich motif in the SeV cap methylation. This study is the first extensive sequence analysis of the L protein domain VI in the family Paramyxoviridae, and it confirms structural and functional similarity of this domain across different families of the order Mononegavirales.     

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.     

Synthesis and transport of protein components of Sendai virus

Summary A method for labeling antibodies with p-aminophenylmercurylacetate (pAPMA) was applied to the study of the synthesis and transport of the S-antigen of Sendai virus in cells in tissue cultures. It was shown that synthesis of S-antigen begins in nucleoli 3–4 hours after infection and as it accumulates it is transported through the nucleus into the cytoplasm. Mature virus particles appear at the edge of the cell wall in 9–12 hours after infection. The evidence given indicates that the inner component of Sendai virus (ribonucleoprotein) is formed in the nucleolus. Some peculiarities of messenger RNA of Sendai virus are discussed.     

The properties of recombinant Sendai virus having the P gene of Sendai virus pi strain derived from BHK cells persistently infected with Sendai virus

We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38°C than at 32°C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38°C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38°C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-β at 32 and 38°C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.Machiko Nishio and Ai Nagata have contributed equally.     

Crystallization of Sendai virus HN protein complexed with monoclonal antibody Fab fragments

The hemagglutinin-neuraminidase (HN) protein of Sendai virus has been isolated from virus particles in a biologically active soluble form after removal by proteolytic digestion of the hydrophobic amino-terminal anchor sequence (S. D. Thompson, W. G. Laver, K. G. Murti, A. Portner, J. Virol., 62, 4653-4660, 1988). The soluble HN exists as both dimers and tetramers, and crystallization trials with each of these forms have so far yielded amorphous material. Dimers complexed with Fab fragments of a monoclonal antibody formed long needle crystals. So far, these are not suitable for X-ray diffraction analysis but the results suggest that HN molecules from paramyxoviruses, even if not crystallizable, may, when complexed with Fab fragments, in some cases yield crystals suitable for X-ray diffraction analysis.