Detection of small RNAs generated during early infection of human HEK 293 cells by the alphavirus Sindbis virus
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE46254 | GEO | 2013/12/01
SECONDARY ACCESSION(S): PRJNA198469
REPOSITORIES: GEO
Similar Datasets
Project description:A time course of infection of the alphavirus Sindbis virus (SINV) was used to investigate the presence of viral specific vsRNA and the changes in miRNAs profiles in human embryonic kidney 293 cells (HEK293) by high throughput DNA sequencing. Deep sequencing of small RNAs early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral specific RNAs (vsRNAs) , with a random uniform distribution not typical of Dicer products, suggesting they arise from non-specific degradation. Sequencing showed little variation of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed insignificant modulation by Northern blot analysis. RNA was isolated from mock infected and SINV inoculated HEK 293 cells at 4hpi and 6hpi cDNA libraries were generated for the small RNA (sRNA) content of the cells and sequenced using Illumina GA II, which yielded between 29.1M and 30.5M reads per sample
2013-12-01 | E-GEOD-46254 | biostudies-arrayexpress
Project description:Small RNAs play a critical role in host-pathogen interaction. In insects, for instance, small RNA-mediated silencing or RNA interference (RNAi) represents the main antiviral defense system. However, the antiviral role of RNAi has not been clearly proven in higher vertebrates. On the contrary, it is well established that the cell response relies on the recognition of viral RNAs by host pattern recognition receptors (PRR) to trigger the activation of the interferon pathway. Based on this evidence, we wished to contribute to this research field by identifying and characterizing small non-coding RNAs produced in mammalian cells upon RNA virus infection. We focused on Sindbis virus (SINV), the prototypical arbovirus, which by definition, is able to infect both vertebrate hosts and invertebrate vectors and triggers the interferon pathway or RNAi, respectively. Taking advantage of large scale sequencing, we cloned and sequenced small RNAs from both mock and SINV-infected mammalian cells (HEK 293 and VERO). We identified a novel population of viral small RNAs (vsRNAs) that accumulate as 20 to 30 nt species during infection. We assessed that this viral small RNA population is modified in 3'end and derived from the activation of the cellular antiviral endoribonuclease RNaseL. Altogether our results indicate a potential role for the SINV-derived small RNAs in the host defense mechanism.
2013-11-19 | E-GEOD-48831 | biostudies-arrayexpress
Project description:RNA interference (RNAi) functions as an antiviral immune response in plants and invertebrates, whereas mammalian RNAi response has been found so far only in undifferentiated cells and in differentiated cells inactive in interferon (IFN) system or in infections with viruses disabling viral suppressors of RNAi (VSRs), thereby leading to question the physiological importance of the RNAi pathway in mammals. Here, we identified that wild-type Semliki Forest virus (SFV), a prototypic alphavirus, triggered the Dicer-dependent production of abundant viral (v)siRNAs in different mammalian somatic cells in the presence of VSR. These vsiRNAs were produced from viral dsRNA replicative intermediates, almost exclusively located at the 5’ termini of the viral genome, and loaded into AGO, and they were fully active in slicing cognate viral RNAs. Besides, Sindbis virus, another alphavirus, also induced vsiRNA generation in mammalian somatic cells. AGO2 deficiency increased SFV and SINV replication, while enoxacin, a known RNAi enhancer that functions at post steps of siRNA production, efficiently reduced viral replication. The nucleotide sequence at the 5’ termini of SFV and SINV genome is conserved among the Old World alphaviruses, and mutating the conserved sequences resulted in the recombinant SFV being deficient in vsiRNA production and irresponsive to antiviral RNAi. SFV infection also enabled the production of abundant vsiRNAs and antiviral RNAi in IFN-competent adult mice, and importantly, enhanced RNAi by enoxacin protected adult mice from lethal SFV challenge and reduced the virus-induced neuropathogenesis in the central neuron system. Overall, our findings provide evidence that mammalian antiviral RNAi is active in differentiated cells and adult mice with intact IFN response even in the presence of VSR and present a therapeutic strategy against alphaviruses that include many important emerging and reemerging human pathogens.
2022-10-04 | GSE214453 | GEO
Project description:Arthropod-borne viruses, such as the members of genus Alphavirus, are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduces a robust viral RNA:protein discovery method to elucidate the Sindbis virus (SINV) RNA:Protein host interface. Cross-Link Assisted mRNP Purification (CLAMP) assessment reveals an extensive array of host/pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of Protein:vRNA interaction via a CLIP-seq approach and assessed the consequences of the RNA:protein binding event of hnRNP K, hnRNP I, and hnRNP M in regards to viral infection. Herein we demonstrate that mutation of the prioritized hnRNP:vRNA interaction sites effectively disrupted the hnRNP:vRNA interaction. Correlating with disrupted hnRNP:vRNA binding, SINV growth kinetics were reduced relative to wild type parental viral infections in a vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics was found to be reduced vRNA accumulation and dysregulated structural gene expression. Collectively, this study further defines the scope and importance of the alphavirus host/pathogen vRNA:protein interactions.
2018-01-02 | GSE103693 | GEO
Project description:Alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. While the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. Mutational analyses of the cytoplasmic viral RNA-capsid interaction sites revealed a functional role for capsid binding early in infection. Interaction site mutants exhibited decreased viral growth kinetics; however, this defect was not a function of decreased particle production. Rather mutation of the cytoplasmic capsid-RNA interaction sites negatively affected the functional capacity of the incoming viral genomic RNAs leading to decreased infectivity. Furthermore, cytoplasmic capsid interaction site mutants are attenuated in a murine model of neurotropic alphavirus infection. Collectively, the findings of this study indicate that the identified cytoplasmic interactions of the viral capsid protein and genomic RNA, while not essential for particle formation, are necessary for genomic RNA function early during infection. This previously unappreciated role of capsid protein during the alphaviral replication cycle also constitutes a novel virulence determinant.
2017-06-19 | GSE99879 | GEO
Project description:The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 108 pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had twenty-five to forty-fold increases in transcript levels in virus infected mosquitoes at 4 days post infection . These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of C. elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and JNK immune cascades as a response to viral infection
2005-08-01 | E-MEXP-381 | biostudies-arrayexpress
Project description:We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito, Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3'UTRs. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific qPCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes. Examination of endo-siRNA production in Aedes aegypti mosquitoes
2012-04-01 | E-GEOD-35161 | biostudies-arrayexpress
Project description:The DEAD-box ATP-dependent RNA helicases DDX5 and DDX17 play a role in many aspects of cellular RNA biology, including metabolism, translation, splicing, transcription regulation, ribosome biogenesis, mRNA nuclear export, and miRNA processing. Moreover, both RNA helicases were found to either promote or inhibit viral replication upon several RNA virus infections. Here we show that DDX5 depletion by siRNA or CRISPR/Cas9 has a negative impact on Sindbis virus (SINV) infection at the viral protein, RNA and infectious particle level. Moreover, we demonstrate that DDX5 which is predominately nuclear in uninfected conditions, re-localizes to the cytoplasm upon infection where it interacts with the viral RNA and with the SINV capsid protein. Furthermore, proteomic analysis of DDX5 interactome in mock and SINV infected HCT116 cells confirmed its interaction with DDX17 and identified PNPT1 as a new DDX5 partner. Of note, while PNPT1 localization remains mostly unchanged in mock and infected cells, DDX17 re-localizes to the cytoplasm with DDX5 upon SINV infection and interacts with SINV capsid protein. Finally, depletion of DDX17 further reduces SINV infection in a DDX5-depleted background suggesting a cumulative proviral effect of DDX5 and 17 proteins on SINV.
2024-05-22 | PXD044867 | Pride
Project description:We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito, Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3'UTRs. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific qPCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes.
2012-04-01 | GSE35161 | GEO
Project description:Viruses are obligate intracellular parasites, which depend on the host cellular machineries to replicate their genome and complete their infectious cycle. Long double stranded (ds)RNA is a common viral by-product originating during RNA virus replication, which is universally sensed as a danger signal to trigger the antiviral response. As a result, viruses hide dsRNA intermediates into viral replication factories and have evolved strategies to hijack cellular proteins for their benefit. The characterization of the host factors associated to viral dsRNA and involved in viral replication remains a major challenge to develop new antiviral drugs against RNA viruses. Here, we performed anti-dsRNA immunoprecipitation followed by Mass Spectrometry to fully characterize the dsRNA interactome in Sindbis virus (SINV) infected human HCT116 cells. Among the validated factors, we characterized SFPQ (Splicing factor, proline-glutamine rich) as a new dsRNA-associated factor upon SINV infection. We proved that SFPQ is able to directly bind dsRNAs in vitro, that SFPQ association to dsRNA is independent on single-stranded (ss)RNA flanking regions in vivo and that it is able to bind the viral genome upon infection. Furthermore, we showed that either knock-down or knock-out of SFPQ reduced SINV infection in human HCT116 and SKNBE cells, suggesting that SFPQ could enhance viral replication. Overall, this study not only represents a resource to further study SINV dsRNA-associated factors upon infection but also identifies SFPQ as a new proviral dsRNA binding protein.
2024-05-22 | PXD024554 | Pride