RESUMO
ABSTRACTEnterovirus A71 (EV-A71) can cause hand, foot and mouth disease with neurological and systemic complications, most frequently affecting children and infants. We describe a cis-acting replication element (cre) with a conserved stem-loop structure within the EV-A71 2C-coding region. By site-directed mutagenesis and reverse genetics using the EV-A71 full-length genome and the EV-A71 replicon containing the firefly luciferase reporter gene in place of the P1 region, the stem-loop structure and the AAACA in the loop of the cre were confirmed to be required for the EV-A71 replication phenotype. EV-A71 genomes containing a mutation at the first or third A residue of AAACA could not be recovered. Insertion of a wild-type cre from EV-A71 or poliovirus in the 5'UTR led to successful recovery of the replication of nonviable mutants. Furthermore, the cre mutants showed lower binding capacity with the host cellular factor IGF2BP2, knockdown of which resulted in a significant decrease in EV-A71 production. All the available evidence shows the location independence but functional importance of the interaction of the cre with the cellular host for efficient production of EV-A71, contributing to the growing body of knowledge regarding picornavirus cres.
Assuntos
Enterovirus Humano A/genética , Genoma Viral/genética , Sequências Repetitivas Dispersas/genética , Conformação de Ácido Nucleico , RNA Viral/genética , Replicação Viral/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Enterovirus Humano A/crescimento & desenvolvimento , Infecções por Enterovirus/virologia , Doença de Mão, Pé e Boca/virologia , Humanos , Integrases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Células VeroRESUMO
Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has caused periodic infection outbreaks in children in the Asia-Pacific region. In order to describe the largely unknown life cycle of EV71, the molecular basis of its virus-host interactions must first be determined. The 5' untranslated region of EV71 contains a cloverleaf-like structure and internal ribosomal entry site (IRES), which play an important role in transcription and translation of viral protein. We found that polypyrimidine tract-binding protein 1 (PTB) bound to the IRES of EV71. RNA recognition motifs 1 and 2 of PTB were responsible for its binding to the EV71 IRES. Moreover, PTB protein was shuttled from nucleus to cytoplasm after EV71 infection. Additionally, IRES activity and viral protein production were inhibited by PTB knockdown. These results suggest that PTB interacts with the EV71 IRES, and positively regulates viral protein translation.
Assuntos
Enterovirus Humano A/genética , Interações entre Hospedeiro e Microrganismos/genética , Sítios Internos de Entrada Ribossomal/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , Regiões 5' não Traduzidas/genética , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , RNA Viral , Proteínas Virais/metabolismo , Replicação ViralRESUMO
In a yeast two-hybrid screen, we identified the microtubule-destabilizing protein SCG10 as a potential effector protein of BRI3. The association was verified using GST pull-down, Co-IP, and their perinuclear co-localization. The analysis of in vitro microtubule polymerization/depolymerization showed that the binding of BRI3 to SCG10 effectively blocked the ability of SCG10 to induce microtubule disassembly, as determined by turbidimetric assays. In intact PC12 cells, BRI3 exhibited the ability to stabilize the microtubule network and attenuate the microtubuledestabilizing activity of SCG10. Furthermore, co-expression of BRI3 with SCG10 attenuated SCG10-mediated PC12 cell neurite outgrowth induced by NGF. These results identify a novel connection between a neuron-specific BRI protein and the cytoskeletal network, suggesting possible roles of BRI3 in the process of neuronal differentiation.