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1.
J Biol Chem ; 294(49): 18742-18755, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666336

RESUMO

The centrosome is a cytoplasmic nonenveloped organelle functioning as one of the microtubule-organizing centers and composing a centriole center surrounded by pericentriolar material (PCM) granules. PCM consists of many centrosomal proteins, including PCM1 and centrosomal protein 131 (CEP131), and helps maintain centrosome stability. Zika virus (ZIKV) is a flavivirus of the family Flaviviridae whose RNA and viral particles are replicated in the cytoplasm. However, how ZIKV interacts with host cell components during its productive infection stage is incompletely understood. Here, using several primate cell lines, we report that ZIKV infection disrupts and disperses the PCM granules. We demonstrate that PCM1- and CEP131-containing granules are dispersed in ZIKV-infected cells, whereas the centrioles remain intact. We found that ZIKV does not significantly alter cellular skeletal proteins, and, hence, these proteins may not be involved in the interaction between ZIKV and centrosomal proteins. Moreover, ZIKV infection decreased PCM1 and CEP131 protein, but not mRNA, levels. We further found that the protease inhibitor MG132 prevents the decrease in PCM1 and CEP131 levels and centriolar satellite dispersion. Therefore, we hypothesized that ZIKV infection induces proteasomal PCM1 and CEP131 degradation and thereby disrupts the PCM granules. Supporting this hypothesis, we show that ZIKV infection increases levels of mind bomb 1 (MIB1), previously demonstrated to be an E3 ubiquitin ligase for PCM1 and CEP131 and that ZIKV fails to degrade or disperse PCM in MIB1-ko cells. Our results imply that ZIKV infection activates MIB1-mediated ubiquitination that degrades PCM1 and CEP131, leading to PCM granule dispersion.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Infecção por Zika virus/metabolismo , Animais , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HEK293 , Humanos , Immunoblotting , Ubiquitina-Proteína Ligases/genética , Células Vero , Zika virus , Infecção por Zika virus/genética
2.
BMC Infect Dis ; 17(1): 239, 2017 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-28359304

RESUMO

BACKGROUND: Two lineages of Zika virus (ZIKV) have been classified according to the phylogenetic analysis: African and Asian lineages. It is unclear whether differences exist between the two strains in host cell permissiveness, this information is important for understanding viral pathogenesis and designing anti-viral strategies. METHODS: In the present study, we comparatively studied the permissive spectrum of human cells for both the African (MR766) and Asian strains (PRVABC59) using an RNA in situ hybridization (RISH) to visualize RNA replication, an immunofluorescence technology, and a western blot assay to determine viral protein production, and a real-time RT-PCR to examine viral RNA multiplication level. The experiments were undertaken in the condition of cell culture. RESULTS: We identified several human cell lines, including fibroblast, epithelial cells, brain cells, stem cells, and blood cells that are susceptible for the infection of both Asian and African strains. We did not find any differences between the MR766 and the PRVABC59 in the permissiveness, infection rate, and replication modes. Inconsistent to a previous report (Hamel et al. JVI 89:8880-8896, 2015), using RISH or real-time RT-PCR, we found that human foreskin fibroblast cells were not permissive for ZIKV infection. Instead, human lung fibroblast cells (MRC-5) were fully permissive for ZIKV infection. Surprisingly, a direct interaction of ZIKV RNA with envelop (E) protein (a structure protein) was demonstrated by an RNA chromatin immunoprecipitation (ChIP) assay. Three binding sites were identified in the ZIKV RNA genome for the interaction with the E protein. CONCLUSION: Our results imply that the E protein may be important for viral RNA replication, and provide not only the information of ZIKV permissiveness that guides the usage of human cells for the ZIKV studies, but also the insight into the viral RNA-E protein interaction that may be targeted for intervention by designing small molecule drugs.


Assuntos
RNA Viral , Proteínas do Envelope Viral/genética , Zika virus/genética , Zika virus/patogenicidade , África , Ásia , Western Blotting , Linhagem Celular , Marcadores Genéticos , Humanos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/genética
3.
Artigo em Inglês | MEDLINE | ID: mdl-38050490

RESUMO

Objective: Dengue viruses (DENV) and Zika viruses (ZIKV) are transmitted from human to human or from non-human primates to humans by mosquito biting, so the viral interaction with mosquito cells is one key step within the viral life cycle. Therefore, our objective is to know how DENV or ZIKV interacts with mosquito cells. Methods: Immunofluorescence assay and a direct visualization system are combined to monitor the syncytial or congregative effects of DENVs and ZIKVs on C6/36 cells. we studied the cytopathic effects of DENVs and ZIKVs on the mosquito cells, C6/36 which are widely used in the laboratory for the infections of DENV and ZIKV. Results: Our results show that all strains of DENV-1 and DENV-2, most DENV-4 and some DENV-3 strains caused syncytial effects on C6/36 cells, while some DENV-3 and DENV-4 strains, and all the tested ZIKV strains caused cell congregation after infection but no cell fusion. In addition, we detected a range of pH environments from 6.0 to 8.0 that support the virus-caused cell fusion and figured out that the optimal pH condition is 7.5 at which the viral production is also the best. Furthermore, viral replication may be required for DENV's syncytial effects on C6/36 cells because the UV-inactivated virus failed to cause cell fusion. Conclusion: Syncytial and congregative effects of DENV and ZIKV on the Aedes albopictus cells differ among the viral strains. Syncytial effects of DENV on C6/36 are important for viral replication.

4.
Gene ; 628: 117-128, 2017 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-28720531

RESUMO

Zika virus (ZIKV) encodes a precursor protein (also called polyprotein) of about 3424 amino acids that is processed by proteases to generate 10 mature proteins and a small peptide. In the present study, we characterized the chemical features, suborganelle distribution and potential function of each protein using Flag-tagged protein expression system. Western blot analysis revealed the molecular weight of the proteins and the polymerization of E, NS1, and NS3 proteins. In addition, we performed multi-labeled fluorescent immunocytochemistry and subcellular fractionation to determine the subcellular localization of these proteins in host cells. We found that 1) the capsid protein colocalizes with 3 different cellular organelles: nucleoli, Golgi apparatus, and lipid droplet; NS2b and NS4a are associated with the Golgi apparatus; 2) the capsid and NS1proteins distribute in both cytoplasm and nucleus, NS5 is a nuclear protein; 3) NS3 protein colocalizes with tubulin and affects Lamin A; 4) Envelope, PrM, and NS2a proteins co-localize with the endoplasmic reticulum; 5) NS1 is associated with autophagosomes and NS4b is related to early endosome; 6) NS5 forms punctate structures in the nucleus that associate with splicing compartments shown by SC35, leading to reduction of SC35 protein level and trafficking of SC35 from the nucleus to the cytoplasm. These data suggest that ZIKV generates 10 functional viral proteins that exhibit distinctive subcellular distribution in host cells.


Assuntos
Proteínas Virais/genética , Zika virus/genética , Animais , Autofagia , Nucléolo Celular/virologia , Núcleo Celular/virologia , Chlorocebus aethiops , Clonagem Molecular , Citoplasma/virologia , Retículo Endoplasmático/virologia , Endossomos/virologia , Genes Virais , Vetores Genéticos , Complexo de Golgi/virologia , Células HEK293 , Humanos , Lamina Tipo A/metabolismo , Transfecção , Tubulina (Proteína)/metabolismo , Células Vero , Proteínas não Estruturais Virais/genética , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/genética
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