RESUMEN
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.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Niño , Humanos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Cromatografía Liquida , Unión Proteica , Factores de Empalme de ARN/metabolismo , Espectrometría de Masas en Tándem , Proteínas no Estructurales Virales/genética , GTP Ciclohidrolasa/metabolismoRESUMEN
Zika virus (ZIKV) targets the neural progenitor cells (NPCs) in brain during intrauterine infections and consequently causes severe neurological disorders, such as microcephaly in neonates. Although replicating in the cytoplasm, ZIKV dysregulates the expression of thousands of host genes, yet the detailed mechanism remains elusive. Herein, we report that ZIKV encodes a unique DNA-binding protein to regulate host gene transcription in the nucleus. We found that ZIKV NS5, the viral RNA polymerase, associates tightly with host chromatin DNA through its methyltransferase domain and this interaction could be specifically blocked by GTP. Further study showed that expression of ZIKV NS5 in human NPCs markedly suppressed the transcription of its target genes, especially the genes involved in neurogenesis. Mechanistically, ZIKV NS5 binds onto the gene body of its target genes and then blocks their transcriptional elongation. The utero electroporation in pregnant mice showed that NS5 expression significantly disrupts the neurogenesis by reducing the number of Sox2- and Tbr2-positive cells in the fetal cortex. Together, our findings demonstrate a molecular clue linking to the abnormal neurodevelopment caused by ZIKV infection and also provide intriguing insights into the interaction between the host cell and the pathogenic RNA virus, where the cytoplasmic RNA virus encodes a DNA-binding protein to control the transcription of host cell in the nuclei.
Asunto(s)
Infección por el Virus Zika , Virus Zika , Humanos , Femenino , Embarazo , Animales , Ratones , Cromatina/genética , Virus Zika/genética , Infección por el Virus Zika/genética , ADN , ARN Polimerasas Dirigidas por ADN/genética , Transcripción GenéticaRESUMEN
Duck Tembusu virus (TMUV) belongs to the flavivirus genus whose genome replication involved in capping and RNA synthesis dominating by nonstructural protein 5 (NS5). Flaviviral replication has been well documented to occur in the cytoplasm, but the effect of NS5 to gain access to the nucleus remains controversial. Here, TMUV NS5 was observed to localize within the cytoplasm of transfected and infected cells and co-localized with the endoplasmic reticulum. We introduced two arginine mutations into the N390 and Q392 (N390R and Q392R) of the NS5 bipartite nuclear localization sequence (α/ßNLS) and designated that mutagenesis as NS5NLSmut, which has shown the ability to access the nucleus and hence attenuates viral replication and production in vitro. Additionally, there was no significant difference between the recovered wild-type TMUV (rTMUV-WT) and engineered mutant (rTMUV-NS5NLSmut) on plaque morphology, survival rate of infected duck embryos or virus copies in tissues. Considering that NS5NLSmut is mainly located in the cytoplasm of rTMUV-NS5NLSmut infected cells at the early stage of infection. We further confirmed that NS5NLSmut attenuated its interaction with nonstructural NS2B-NS3 (NS2B3) following transfection and infection. Meanwhile, the rTMUV-NS5NLSmut tended to stimulate more interferon beta (IFNß) than rTMUV-WT. However, preliminary study on transient NS5 and NS5NLSmut detected the same levels of IFNß mRNA mediated by RIG-I detection of NS5 RNA polymerase activity in cell. In summary, these results provide further insights into the relationship between the viral property and subcellular localization of flavivirus NS5 in terms of the NS5-NS2B3 interaction.
Asunto(s)
Flavivirus , Proteínas no Estructurales Virales , Replicación Viral , Animales , Arginina/genética , Núcleo Celular/virología , Flavivirus/fisiología , Interferón beta/genética , Mutación , Transporte de Proteínas , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Zika virus (ZIKV) infection could disrupt neurogenesis and cause microcephaly in neonates by targeting neural progenitor cells (NPCs). The tumor suppressor p53-mediated cell cycle arrest and apoptotic cell death have been suggested to be activated upon ZIKV infection, yet the detailed mechanism is not well understood. In the present study, we investigated the effects of ZIKV-encoded proteins in the activation of p53 signaling pathway and found that, among the ten viral proteins, the nonstructural protein 5 (NS5) of ZIKV most significantly activated the transcription of p53 target genes. Using the immunoprecipitation-coupled mass spectrometry approach, we identified that ZIKV-NS5 interacted with p53 protein. The NS5-p53 interaction was further confirmed by co-immunoprecipitation and GST pull-down assays. In addition, the MTase domain of NS5 and the C-terminal domain of p53 were mapped to be responsible for the interaction between these two proteins. We further showed that ZIKV-NS5 was colocalized with p53 and increased its protein level in the nuclei and able to prolong the half-life of p53. Furthermore, lentivirus-mediated expression of ZIKV-NS5 in hNPCs led to an apparent cell death phenotype. ZIKV-NS5 promoted the cleavage of PARP1 and significantly increased the cell apoptosis of hNPCs. Taken together, these findings revealed that ZIKV-NS5 is a previously undiscovered regulator of p53-mediated apoptosis in hNPCs, which may contribute to the ZIKV-caused abnormal neurodevelopment.
Asunto(s)
Apoptosis , Células-Madre Neurales , Proteína p53 Supresora de Tumor , Proteínas no Estructurales Virales , Infección por el Virus Zika , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/virología , Proteína p53 Supresora de Tumor/genética , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Virus Zika/metabolismoRESUMEN
For many viruses, RNA is the holder of genetic information and serves as the template for both replication and translation. While host and viral proteins play important roles in viral decision-making, the extent to which viral RNA (vRNA) actively participates in translation and replication might be surprising. Here, the focus is on flaviviruses, which include common human scourges such as dengue, West Nile, and Zika viruses, from an RNA-centric viewpoint. In reviewing more recent findings, an attempt is made to fill knowledge gaps and revisit some canonical views of vRNA structures involved in replication. In particular, alternative views are offered on the nature of the flaviviral promoter and genome cyclization, and the feasibility of refining in vitro-derived models with modern RNA probing and sequencing methods is pointed out. By tracing vRNA structures from translation through encapsidation, a dynamic molecule closely involved in the self-regulation of viral replication is revealed.