Sendai virus causes a rise in intracellular free Ca2+ before cell fusion.
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ABSTRACT:
SUBMITTER: Hallett MB
PROVIDER: S-EPMC1158639 | biostudies-other | 1982 Sep
REPOSITORIES: biostudies-other
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Project description:BackgroundParamyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.Methodology/principal findingsTo analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Conclusions/significanceCollectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses.
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Project description:Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion-mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206-an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.
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Project description:Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5-7?mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10-13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.
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Project description:Glioblastoma multiforme (GM), the most frequent primary malignant brain tumor, is highly invasive due to the expression of proteases, including urokinase-type plasminogen activator (uPA). Here, we show the potential of our new and powerful recombinant Sendai virus (rSeV) showing uPA-specific cell-to-cell fusion activity [rSeV/dMFct14 (uPA2), named "BioKnife"] for GM treatment, an effect that was synergistically enhanced by arming BioKnife with the interferon-? (IFN-?) gene. BioKnife killed human GM cell lines efficiently in a uPA-dependent fashion, and this killing was prevented by PA inhibitor-1. Rat gliosarcoma 9L cells expressing both uPA and its functional receptor uPAR (9L-L/R) exhibited high uPA activity on the cellular surface and were highly susceptible to BioKnife. Although parent 9L cells (9L-P) were resistant to BioKnife and to BioKnife expressing IFN-? (BioKnife-IFN?), cell-cell fusion of 9L-L/R strongly facilitated the expression of IFN-?, and in turn, IFN-? significantly accelerated the fusion activity of BioKnife. A similar synergy was seen in a rat orthotopic brain GM model with 9L-L/R in vivo; therefore, these results suggest that BioKnife-IFN? may have significant potential to improve the survival of GM patients in a clinical setting.
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Project description:Enveloped viruses enter host cells through membrane fusion and the cells in turn alter their shape to accommodate components of the virus. However, the role of nonmuscle myosin II of the actomyosin complex of host cells in membrane fusion is yet to be understood. Herein, we show that both (-) blebbistatin, a specific inhibitor of nonmuscle myosin II (NMII) and small interfering RNA markedly augment fusion of Sendai virus (SeV), with chinese hamster ovary cells and human hepatocarcinoma cells. Inhibition of RLC phosphorylation using inhibitors against ROCK, but not PKC and MRCK, or overexpression of phospho-dead mutant of RLC enhances membrane fusion. SeV infection increases cellular stiffness and myosin light chain phosphorylation at two hour post infection. Taken together, the present investigation strongly indicates that Rho-ROCK-NMII contractility signaling pathway may provide a physical barrier to host cells against viral fusion.
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Project description:Neuronal communication is mediated by Ca(2+)-triggered fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. Synaptotagmin I functions as a Ca(2+) sensor that regulates exocytosis, whereas soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins in the vesicle and target membrane assemble into complexes that directly catalyze bilayer fusion. Here we report that, before the Ca(2+) trigger, synaptotagmin interacts with SNARE proteins in the target membrane to halt SNARE complex assembly at a step after donor vesicles attach, or dock, to target membranes. This results in fusion complexes that, when subsequently triggered by Ca(2+), drive rapid, highly efficient lipid mixing. Ca(2+)-independent interactions with SNAREs also predispose synaptotagmin to selectively penetrate the target membrane in response to Ca(2+); we demonstrate that Ca(2+)-synaptotagmin must insert into the target membrane to accelerate SNARE-catalyzed fusion. These findings demonstrate that Ca(2+) converts synaptotagmin from a clamp to a trigger for exocytosis.
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Project description:Induced pluripotent stem cells (iPSCs) are potentially valuable cell sources for disease models and future therapeutic applications; however, inefficient generation and the presence of integrated transgenes remain as problems limiting their current use. Here, we developed a new Sendai virus vector, TS12KOS, which has improved efficiency, does not integrate into the cellular DNA, and can be easily eliminated. TS12KOS carries KLF4, OCT3/4, and SOX2 in a single vector and can easily generate iPSCs from human blood cells. Using TS12KOS, we established iPSC lines from chimpanzee blood, and used DNA array analysis to show that the global gene-expression pattern of chimpanzee iPSCs is similar to those of human embryonic stem cell and iPSC lines. These results demonstrated that our new vector is useful for generating iPSCs from the blood cells of both human and chimpanzee. In addition, the chimpanzee iPSCs are expected to facilitate unique studies into human physiology and disease.
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Project description:In paramyxovirus transcription, viral RNA polymerase synthesizes each monocistronic mRNA by recognizing the gene start (S) and end (E) signals flanking each gene. These signal sequences are well conserved in the virus family; nevertheless, they do exhibit some variations even within a virus species. In Sendai virus (SeV) Z strain, the E signals are identical for all six genes but there are four (N, P/M/HN, F, and L) different S signals with one or two nucleotide variations. The significance of these variations for in vitro and in vivo replication has been unknown. We addressed this issue by SeV reverse genetics. The luciferase gene was placed between the N and P gene so that recombinant SeVs expressed luciferase under the control of each of the four different S signals. The S signal for the F gene was found to drive a lower level of transcription than that of the other three, which exhibited comparable reinitiation capacities. The polar attenuation of SeV transcription thus appeared to be not linear but biphasic. Then, a mutant SeV whose F gene S signal was replaced with that used for the P, M, and HN genes was created, and its replication capability was examined. The mutant produced a larger amount of F protein and downstream gene-encoded proteins and replicated faster than wild-type SeV in cultured cells and in embryonated eggs. Compared with the wild type, the mutant virus also replicated faster in mice and was more virulent, requiring a dose 20 times lower to kill 50% of mice. On the other hand, the unique F start sequence as well as the other start sequences are perfectly conserved in all SeV isolates sequenced to date, including highly virulent fresh isolates as well as egg-adapted strains, with a virulence several magnitudes lower than that of the fresh isolates. This moderation of transcription at the F gene may therefore be relevant to viral fitness in nature.
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