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SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

ABSTRACT: We performed a transcriptomic analysis of H293T cells expressing individual SARS-CoV2 proteins. Overexpression of Nsp14, a protein known to be involved in viral RNA replication, provoked a dramatic remodeling of the transcriptome, altering the expression of ≈4000 RNAs (3'RNAseq). These transcriptome changes strongly resemble the ones observed following SARS-CoV-2 infection. We performed a timecourse 3'RNA seq experiment and detected genes related to NFkB activation as deregulated soon after the transfection. We showed that the effect does not depend by the presence of the co-factor Nsp10 (total RNA seq) or by the exonuclease activity of Nsp14 (3' RNA seq of Nsp14 D90A/G92A and Nsp14D273 mutants). Finally we saw that the effect are mediated by the cellular enzyme IMPDH2, since treatment with MPA partially rescued the effect induced by Nsp14OE (3'RNA seq).

ORGANISM(S): Homo sapiens

PROVIDER: GSE179251 | GEO | 2022/03/11

REPOSITORIES: GEO

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Project description:Pathogenic mechanisms underlying severe SARS-CoV2 infection remain largely unelucidated. High-throughput sequencing technologies that capture genome and transcriptome information are key approaches to gain detailed mechanistic insights from infected cells. These techniques readily detect both pathogen and host-derived sequences, providing a means of studying host-pathogen interactions. Recent studies have reported the presence of host-virus chimeric (HVC) RNA in RNA-seq data from SARS-CoV2 infected cells and interpreted these findings as evidence of viral integration in the human genome as a pathogenic mechanism. Since SARS-CoV2 is a positive sense RNA virus that replicates in the cytoplasm it does not have a nuclear phase in its life cycle, so it is biologically unlikely to be in a location where splicing events could result in genome integration. Here, we investigated the biological authenticity of HVC events. In contrast to true biological events, e.g. mRNA splicing and genome rearrangement events, which generate reproducible chimeric sequencing fragments across different biological isolates, we found that HVC events across >100 RNA-seq libraries from patients with COVID-19 and SARS-CoV2 infected cell lines, were highly irreproducible. RNA-seq library preparation is inherently error-prone due to random template switching during reverse transcription of RNA to cDNA. By counting chimeric events observed when constructing an RNA-seq library from human RNA and spike-in RNA from an unrelated species, such as fruit-fly, we estimated that ~1% of RNA-seq reads are artifactually chimeric. In SARS-CoV2 RNA-seq we found that the frequency of HVC events were, in fact, no more frequent than this background “noise”. Finally, we developed a novel experimental approach to enrich SARS-CoV2 sequences from bulk RNA of infected cells. This method enriched viral sequences but did not enrich for HVC events, suggesting that the majority of HVC events are, in all likelihood, artifacts of library construction. In conclusion, our findings indicate that HVC events observed in RNA-sequencing libraries from SARS-CoV2 infected cells are extremely rare and are likely artifacts arising from either random template switching of reverse-transcriptase and/or sequence alignment errors. Therefore, the observed HVC events do not support SARS-CoV2 fusion to cellular genes and/or integration into human genomes.

Project description:The goal of this study was to determine changes in the expression of genes in monoctic myleoid derived suppressor cells (M-MDSC) as a result of SARS CoV2 infection. The study aimed to investigate if M-MDSC are functionally active and inhibit T cell function in response to SARS CoV2 antigens 5 months after first detection of the virus. Methods: Peripheral blood mononuclear cells (PBMC) were collected from CoV2 (-) and CoV2 (+) donors (N=5 each group). M-MDSC were isolated by flow cytometry, and RNA extracted for RNA-seq studies. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the latest version of the human genome (build hg38) using HISAT2. Mapped reads were counted against the human GENCODE annotation (v37) using HT-Seq. The EdgeR library in the R computing environment was used for quality control of the RNA-Seq data, and ComBat-seq method for correction of batch effects. Differential gene expression analysis was conducted using EdgeR. Pathway enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8. Results: An average of 34 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,217 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between CoV2 (+) and CoV2 (-) samples using EdgeR. A total of 188 differentially expressed genes (DEGs) were identified with nominal p-value <0.05; of which 63 were up- and 125 downregulated in CoV2 (+) samples. A total of 12 DEGs were identified with false discovery rate corrected p-value <0.05, of which 2 were up- and 10 downregulated. Pathway enrichment analysis identified pathways involved in immune response and innate immune signaling. Conclusion: The study demonstrated that CoV2 infection modulated the expression of genes involved in immune response and innate immune signaling. Most of the genes remained downregulated even after 5 months of first detection of SARS CoV2.

Project description:The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. Small non-coding RNAs (sncRNAs) are known to play important roles in almost all biological processes. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK‐RNA‐seq, a modified next‐generation sequencing (NGS), we recently found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 SARS-CoV- 2-impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5’-end of tRNAs (tRF5). Using a modified qRT-PCR, which was recently developed to specifically quantify tRF5s by isolating the tRF signals from its corresponding parent tRNA signals, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3’-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.

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SARS-CoV-2 Nsp14 mediates the effects of viral infection on the host cell transcriptome

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