RESUMO
Zika virus (ZIKV), a positive-sense single-stranded RNA virus, causes congenital ZIKV syndrome in children and Guillain-Barré Syndrome (GBS) in adults. ZIKV expresses nonstructural protein 5 (NS5), a large protein that is essential for viral replication. ZIKV NS5 confers the ability to evade interferon (IFN) signalling; however, the exact mechanism remains unclear. In this study, we employed affinity pull-down and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and found that splicing factor 3b subunit 3 (SF3B3) is associated with the NS5-Flag pull-down complex through interaction with NS5. Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene (ISG) expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication. GTP cyclohydrolase I (GCH1) is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis. NS5 upregulates the expression of GCH1 during ZIKV infection. And GCH1 marginally promoted ZIKV replication via the IFN pathway. Additionally, GCH1 expression is related to the regulation of SF3B3. Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels, whereas SF3B3 knockdown increased its levels. These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.
Assuntos
Infecção por Zika virus , Zika virus , Criança , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Cromatografia Líquida , Ligação Proteica , Fatores de Processamento de RNA/metabolismo , Espectrometria de Massas em Tandem , Proteínas não Estruturais Virais/genética , GTP Cicloidrolase/metabolismoRESUMO
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
Chitosan composite rods (CS-Fe(3+)) were prepared via an in situ precipitation method. The relationships among the preparation, structures, and properties of the CS-Fe(3+) composite rods have been investigated. The results of Fourier-transform infrared spectroscopy (FTIR) and core electron X-ray photoelectron spectroscopy (XPS) indicate that the CS and Fe(3+) are coordinated via a chelation mechanism. The content of Fe(3+) in the complex was determined by atomic absorption spectrometry (AAS) and elemental analysis (EA), the results of which suggested that the content of Fe(3+) in the complex can be controlled by the concentration of the ferric salts during coordination. The changes in thermal stability and crystallization properties were measured by thermogravimetric analysis (TGA) and X-ray diffraction (XRD) patterns, respectively. Scanning electron microscopy (SEM) was used to observe the morphological change of the CS-Fe(3+) complex rod. After coordination with Fe(3+), the CS rod had a denser, layered structure. However, the layered structure cannot remain intact when the ratios of -NH(2)/Fe(3+) are 100/15 and 100/20. Moreover, its thermal stability decreased, and its bending strength was improved significantly (from 86 MPa to more than 210 MPa), despite the remarkable decrease in the degree of crystallinity.