Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection

Chang, Jessie; Rawlinson, Daniel; Pitt, Miranda E.; Taiaroa, George; Gleeson, Josie; Zhou, Chenxi; Mordant, Francesca L; Paoli-Iseppi, Ricardo De; Caly, Leon; Purcell, Damian F. J.; Stinear, Timothy P.; Londrigan, Sarah L.; Clark, Michael B.; Williamson, Deborah A; Subbarao, Kanta; Coin, Lachlan

doi:10.1016/j.celrep.2021.109108

Cited by 30 publications

(51 citation statements)

References 39 publications

(42 reference statements)

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“…However, we observed the absence/low expression of ACE2 (< 5 reads per replicate) and TMPRSS2 (< 25 reads per replicate) genes across all our susceptible cell lines. The presence of these transcripts correlated with the viral burden observed in each cell line from our previous study (Data S1) (Chang et al, 2021).…”

Section: Cell-type Specific Changes In Host Gene Expression In Vitro Following Virus Infection Using Long-read Sequencingsupporting

confidence: 60%

“…Publicly available data from our previous work (Chang et al, 2021) in combination with data generated from this study were analysed using Spartan (Meade, Lafayette, Sauter, & Tosello, 2017) et al, 2016) with the default direct RNA parameters '-ax splice -uf -k14 --secondary=no' and for all cDNA datasets '-ax splice -secondary=no'. All data were mapped to the respective combined transcriptome using the following parameters -'-ax map-ont'.…”

Section: Discussionmentioning

confidence: 99%

“…Cell culture and RNA extraction/preparation methods have been described previously (Chang et al, 2021) for Calu-3 (human lung adenocarcinoma epithelial - ATCC HTB-55), Caco-2 (human colorectal adenocarcinoma epithelial - ATCC HTB-37) and Vero (African green monkey kidney epithelial - ATCC CCL-81) cells. For this current study, we additionally cultured A549 (human lung carcinoma epithelial – ATCC CCL-185) cells to supplement our main data, using similar methods.…”

Section: Methodsmentioning

confidence: 99%

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Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

Chang

Gleeson

Rawlinson

et al. 2021

Preprint

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Better methods to interrogate host-pathogen interactions during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infections are imperative to help understand and prevent this disease. Here we implemented RNA-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads to measure differential host gene expression, transcript polyadenylation and isoform usage within various epithelial cell lines permissive and non-permissive for SARS-CoV-2 infection. SARS-CoV-2-infected and mock-infected Vero (African green monkey kidney epithelial cells), Calu-3 (human lung adenocarcinoma epithelial cells), Caco-2 (human colorectal adenocarcinoma epithelial cells) and A549 (human lung carcinoma epithelial cells) were analysed over time (0, 2, 24, 48 hours). Differential polyadenylation was found to occur in both infected Calu-3 and Vero cells during a late time point (48 hpi), with Gene Ontology (GO) terms such as viral transcription and translation shown to be significantly enriched in Calu-3 data. Poly(A) tails showed increased lengths in the majority of the differentially polyadenylated transcripts in Calu-3 and Vero cell lines (up to ~136 nt in mean poly(A) length, padj = 0.029). Of these genes, ribosomal protein genes such as RPS4X and RPS6 also showed downregulation in expression levels, suggesting the importance of ribosomal protein genes during infection. Furthermore, differential transcript usage was identified in Caco-2, Calu-3 and Vero cells, including transcripts of genes such as GSDMB and KPNA2, which have previously been implicated in SARS-CoV-2 infections. Overall, these results highlight the potential role of differential polyadenylation and transcript usage in host immune response or viral manipulation of host mechanisms during infection, and therefore, showcase the value of long-read sequencing in identifying less-explored host responses to disease.

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Section: Cell-type Specific Changes In Host Gene Expression In Vitro Following Virus Infection Using Long-read Sequencingsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

Chang

Gleeson

Rawlinson

et al. 2021

Preprint

Self Cite

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“…The ability to characterise viral transcriptomes with long-read sequencing has been especially useful throughout the COVID19 pandemic. Nanopore sequencing of the SARS-CoV-2 virus revealed the dynamic nature of transcription during its replication cycle (Chang et al, 2021). As well as identifying differential expression of subgenomic mRNA (sgRNA) transcripts during infection, novel sgRNAs containing non-canonical splice junctions were found that may have a role in enhancing viral protein production (Chang et al, 2021).…”

Section: Long Read Profiling Of Splicing In Diseasementioning

confidence: 99%

“…Nanopore sequencing of the SARS-CoV-2 virus revealed the dynamic nature of transcription during its replication cycle (Chang et al, 2021). As well as identifying differential expression of subgenomic mRNA (sgRNA) transcripts during infection, novel sgRNAs containing non-canonical splice junctions were found that may have a role in enhancing viral protein production (Chang et al, 2021). These studies focusing on viral infection highlight how long-read sequencing enables the detection of fulllength isoforms to reveal underlying viral mechanisms and further insight into the viral replication cycle within host human cells.…”

Section: Long Read Profiling Of Splicing In Diseasementioning

confidence: 99%

Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Paoli-Iseppi

Gleeson

Clark

2021

Front. Mol. Biosci.

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Alternative splicing (AS) of RNA is a key mechanism that results in the expression of multiple transcript isoforms from single genes and leads to an increase in the complexity of both the transcriptome and proteome. Regulation of AS is critical for the correct functioning of many biological pathways, while disruption of AS can be directly pathogenic in diseases such as cancer or cause risk for complex disorders. Current short-read sequencing technologies achieve high read depth but are limited in their ability to resolve complex isoforms. In this review we examine how long-read sequencing (LRS) technologies can address this challenge by covering the entire RNA sequence in a single read and thereby distinguish isoform changes that could impact RNA regulation or protein function. Coupling LRS with technologies such as single cell sequencing, targeted sequencing and spatial transcriptomics is producing a rapidly expanding suite of technological approaches to profile alternative splicing at the isoform level with unprecedented detail. In addition, integrating LRS with genotype now allows the impact of genetic variation on isoform expression to be determined. Recent results demonstrate the potential of these techniques to elucidate the landscape of splicing, including in tissues such as the brain where AS is particularly prevalent. Finally, we also discuss how AS can impact protein function, potentially leading to novel therapeutic targets for a range of diseases.

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Secure reversal of immune evasion from refractory NSCLC and highly contagious CoV‐2 mutants by using 3D‐engineered multifunctional biologics

Zhang

Liu

et al. 2023

Bioengineering & Transla Med

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There is an imperative choice to develop a secure feasible strategy to address evasion dynamics of refractory tumors and SARS‐CoV‐2‐variants, while stem cell‐based protocol may be more reliable as its unique ability for resetting multifunctional immunity to address progressive tumor and the constantly‐evolving virus. In this study, spheroid‐embryonoid stem cells from mature somatic cells were engineered as multifunctional biologics (3D‐E/BSC) and inoculated in senile rhesus to identify secure potential against immune‐evasion from viral‐variants. Meanwhile, a cohort of eligible patients with stage IV NSCLC were approved for phase I clinical trials. Subsequently, long‐lasting security and efficacy were validated by primate and clinical trials (p < 0.01) in that it could not only stimulate serological immunity, but also reset core immunity for hosts to address variant evasion after 3D‐E/BSC withdrawal. Particularly, illustrated by single‐cell evolving trajectory, 3D‐E/BSC had securely reset senile thymus of aging hosts to remodel core immunity by rearranging naive rhythm to evolve TRGC2+/JCHAIN+NKT clusters to abolish tumoral and viral evasion dynamics with path‐feedbacks of NSCLC and COVID‐19 simultaneously activated, leading to continuous blockade of breakthrough infection of viral‐mutants and long‐term survival in one‐third of terminal patients without adjuvant required. Our study may pioneer a practical multifunctional strategy to eliminate evasion of SARS‐CoV‐2 variants and refractory NSCLC so as for victims to restart a new life‐equation.

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Transcriptional and epi-transcriptional dynamics of SARS-CoV-2 during cellular infection

Cited by 30 publications

References 39 publications

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection

Isoform Age - Splice Isoform Profiling Using Long-Read Technologies

Secure reversal of immune evasion from refractory NSCLC and highly contagious CoV‐2 mutants by using 3D‐engineered multifunctional biologics

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