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Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein.

ABSTRACT: Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation. The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein. Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively. Our results provide support for a model of viral nucleocapsid axoplasmic transport. Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis.

SUBMITTER: Jacob Y

PROVIDER: S-EPMC102062 | biostudies-literature | 2000 Nov

REPOSITORIES: biostudies-literature

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Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein.

Jacob Y Y Badrane H H Ceccaldi P E PE Tordo N N

Journal of virology 20001101 21

Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3). The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses. The P protein-dynein LC8 interaction was confirmed by col ...[more]

PMID: 11024152

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Project description:Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The phosphoprotein (P) of lyssaviruses (lyssavirus-P) plays multiple roles in virus replication and immune evasion. Lyssavirus-P has been identified as the major type I interferon (IFN-I) antagonist, while the precise site and precise molecular mechanism remain unclear. Herein, we found that substitution of site 179 of lyssavirus-P from serine (Ser) to proline (Pro) impairs its antagonism function of IFN-I by sequence alignment and site mutations. Subsequent studies demonstrated that lyssavirus-P containing S179 specifically interacted with I-kappa B kinase ε (IKKε). Specifically, lyssavirus-P containing S179 interacted simultaneously with the kinase domain (KD) and scaffold dimerization domain (SDD) of IKKε, competing with TNF receptor-associated factor 3 (TRAF3) and IFN regulatory factor 3 (IRF3) for binding with IKKε, leading to the inhibition of IFN production. Furthermore, S179 was involved in the viral pathogenicity of the typical lyssavirus rabies virus in a mouse model. Interestingly, we found that S179 is conserved among most lyssavirus-P and functional for IFN antagonism. Collectively, we identified S179 of lyssavirus-P is essential for IFN-I inhibition, which provides deep insight into the immune evasion strategies of lyssaviruses. IMPORTANCE Interferon (IFN) and the IFN-induced cellular antiviral response constitute the first line of defense against viral invasion. Evading host innate immunity, especially IFN signaling, is the key step required for lyssaviruses to establish infection. In this study, S179 of lyssavirus phosphoprotein (lyssavirus-P) was identified as the key site for antagonizing IFN-I production. Mechanistically, lyssavirus-P containing S179 specifically targets the key kinase IKKε and disrupts its interaction with TRAF3 and IRF3. S179P mutation in the P protein of the typical lyssavirus rabies virus (RABV) attenuated its pathogenicity in a mouse model. Our findings provide deep insight into the immune evasion strategies of lyssaviruses, which is helpful for the development of effective antiviral therapeutics.

Project description:BackgroundDynein Light Chain 1 (LC8) has been shown to pull down tubulin subunits, suggesting that it interacts with microtubules.ResultsLC8 decorates microtubules in vitro and in Drosophila embryos, promotes microtubule assembly, and stabilizes microtubules both in vitro and in tissue-cultured cells.ConclusionLC8 stabilizes microtubules.SignificanceData provide the first evidence of a novel MAP-like function of LC8. Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation.

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Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein.

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Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein.

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