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Detection of small RNAs generated during early infection of human HEK 293 cells by the alphavirus Sindbis virus

ABSTRACT: 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

ORGANISM(S): Homo sapiens

SUBMITTER: Irina Mohorianu

PROVIDER: E-GEOD-46254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Detection of small RNAs generated during early infection of human HEK 293 cells by the alphavirus Sindbis virus

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.

2013-12-01 | GSE46254 | GEO

Identification of RNase L-dependent, 3M-bM-^@M-2-end-modified, viral small RNAs in Sindbis virus-infected mammalian cells

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

DEAD box RNA helicase 5 and 17 are novel host factors for Sindbis virus infection.

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

Determination of dsRNA interactome upon Sindbis virus infection in human cells identifies SFPQ as a critical proviral factor

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

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in the tolerant and susceptible tomato varieties using deep sequencing

Project description:High-throughput sequence analysis of small RNAs of TYLCV-tolerant (containing tolerance gene Ty-1) and susceptible varieties indicated that miRNA-like vsRNAs and secondary vsiRNAs are mainly contributed to hotpots. Interestingly, various characteristics of vsRNAs among each sample are consistent, but in different number of expression; Deep sequencing of degradome provided evidence for the function of vsRNAs-mediated viral transcripts slicing; And bisulfite sequencing PCR suggested the geminivirus DNA methylation induced by vsRNAs. Comparison of the expression quantity and trend of viral DNA, mRNA and vsRNA inferred that the quantity of vsRNAs is significantly corresponding to the expression level of viral mRNA. Nevertheless, vsRNAs had finite effect on inhibition of virus replication and expression. Here, we also speculated that by RDR catalysis of vsRNAs amplification, the tolerance gene Ty-1 may take an effective inhibition of viral transcription at the beginning of TYLCV infection. Examination of 6 different sRNA and 4 degradome library in 2 tomato material. The main result of our paper is distinguish the host plant sRNA and viral-derived sRNA from the .fa files (MMS_21dpi, 30dpi and TY1S_21dpi, 30dpi).

2016-08-22 | E-GEOD-50085 | biostudies-arrayexpress

Characterization and function of Tomato yellow leaf curl virus-derived small RNAs generated in the tolerant and susceptible tomato varieties using deep sequencing

2016-08-22 | GSE50085 | GEO

Cross-species comparative analysis of Dicer proteins during Sindbis virus infection

Project description:The role of RNA silencing as a defense mechanism against viruses remains to be formerly established in mammalian somatic cells. Here, we determined the antiviral properties of human and Drosophila Dicer proteins in a heterologous setup. We expressed human Dicer (hDicer) in Drosophila, and Drosophila Dicer-2 in human cells, and measured the impact on the response to Sindbis virus (SINV) infection. In flies, hDicer presents a low processing activity, but partially rescues a Dcr2 null mutation in flies. Expression of Dicer-2 in HEK293 cells allows the processing of SINV RNA into 21-nt-long small RNAs. Nevertheless, instead of conferring a protective effect against SINV, Dicer-2 expression increases viral replication in HEK293 cells. We present evidence that this effect is due to a competition with the interferon pathway. Our results therefore suggest that adding functionial RNA silencing machinery in IFN-competent differentiated mammalian cells can be detrimental for antiviral defense.

2015-12-28 | GSE62934 | GEO

System-wide profiling of protein-RNA interactions in sindbis infected cells reveals host regulators of infection

Project description:RNA viruses rely on cellular RNA-binding proteins (RBPs) to infect the host cell. However, the repertoire of the cellular RBPs exploited by viruses is largely unknown. Using the ‘RNA interactome capture’ method, we profiled in a proteome-wide scale the RBP-RNA interactions occurring during sindbis virus (SINV) infection. We discover that SINV affects the activity of hundreds of RBPs, essentially rewiring the host RNA-bound proteome. Such alterations do not relate to changes in protein abundance but are rather due to subcellular redistribution of RBPs and pervasive transcriptome remodelling. Strikingly, RBPs stimulated by SINV accumulate in the cytoplasmic compartments where the virus replicates and interact with viral RNA. Perturbation of these RBPs can restrict or promote infection; for example, GEMIN5 binds to key regulatory regions of SINV RNAs and inhibits viral protein expression. Importantly, we show that drug based RBP intervention may have potential as therapeutic strategy against viruses.

2019-02-28 | PXD009789 | Pride

The Identification and Characterization of Sindbis Virus RNA:Host Protein Interactions

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

Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants

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

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