Long-read RNA sequencing identifies important host immune responses during SARS-CoV-2 infection

By Neha MathurReviewed by Benedette Cuffari, M.Sc.Dec 20 2021

In a recent study published on the bioRxiv* preprint server, researchers conduct a comprehensive in vitro analysis of relatively unexplored host immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Continual and concerted exploration efforts are required to devise more effective methodologies to interrogate host-pathogen interactions during SARS-CoV-2 infection, which would help develop more effective diagnostics and therapeutics.

Study: Long-read RNA sequencing identifies polyadenylation elongation and differential transcript usage of host transcripts during SARS-CoV-2 in vitro infection. Image Credit: T-Rex Imagery / Shutterstock.com

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

About the study

The researchers of the present study used ribonucleic acid (RNA)-sequencing (RNA-seq) combined with the Oxford Nanopore Technologies (ONT) long-reads platform to measure differential host gene expression, transcript polyadenylation, and isoform usage for assessing the host immune response to SARS-CoV-2 in vitro. Subsequently, these immune responses were observed at 0-, 2-, 24-, and 48-hours post-infection (hpi) in four epithelial cell lines including Vero, Calu-3, Caco-2, and A549 cells.

Study findings

The poly-A tail is a long chain of adenine nucleotides added to a messenger RNA (mRNA) molecule during RNA processing to increase its stability. The researchers explored poly(A) length and gene expression levels and found that majority of the genes involved in these observations were ribosomal protein genes, such as RPS4X and RPS6.

Previous studies have reported decreased expression of ribosomal proteins in response to SARS-CoV-2 infection due to the effect of global suppression of ribosomal activity initiated by the virus. However, the observed increase in poly(A) lengths in the same transcripts were unexpected, as the elongation of poly(A) tails are indicative of increased stability.

Currently, there is no scientific explanation for this phenomenon. However, it is believed that this may be due to competition between virus-driven expression downregulation and host-driven post-transcriptional regulation for increased mRNA stability.

Next, the researchers observed a substantial increase in the non-mitochondrial median poly(A) tail lengths in Caco-589 2, Calu-3, and Vero cell lines at 24 and 48 hpi, thus suggesting that infection with SARS-CoV-2 may also increase the poly(A) lengths of non-mitochondrial transcripts. Furthermore, as elongated poly(A) tails were observed at 48 hpi in Vero cells, differential polyadenylation may more likely be occurring at later stages of SARS-CoV-2 infection rather than at early stages.

Exploring these results further using tailfindr, the researchers observed poly(A) tail elongation after infection in Calu-3 and Vero cells at 48 hpi. In Calu-3 cells, six genes including RPS12, RPL30, RPS6, RPL13, RPS4X, and RPL10 were involved in viral transcription, thereby indicating that polyadenylation aids in viral mRNA synthesis and is thus needed for replication during SARS-CoV-2 infection.

Interestingly, long non-coding RNAs (lncRNA) and a few mitochondrial transcripts were also observed with an elongated poly(A) length in infected cells compared with control cells. This suggests that protein-coding genes may also be involved in host responses to SARS-CoV-2.

In the past, differential transcript usage has proven useful in studying diseases such as Parkinson’s disease. The researchers observed that in Caco-2, Calu-3, and Vero SARS-CoV-2- susceptible cell lines, processed and retained-intron transcripts were present. Calu-3 48 hpi data revealed the highest number of genes that had undergone differential transcript usage, whereas a retained-intron transcript GSDMB-208 showed differential usage in infected cells compared with control cells.

Furthermore, Karyopherin alpha 2 (KPNA2) transcripts showed differential usage in Calu-3 cells at 48 hpi. These findings suggested that transcripts with differential usage may be involved in pathways contributing to host responses towards viral infection. Hence, the specific activity of each transcript, as opposed to studying their gene-level activity, should be further investigated.

Conclusions

The current study successfully characterized differential expression, polyadenylation, and differential transcript usage of cell lines infected in vitro by SARS-CoV-2. Moreover, it also highlighted the value of long-read sequencing methodologies like short-read RNA-seq in demonstrating the immune responses of four different cell types. These experiments elucidated the pathways associated with infection at different time points, thereby providing novel insights into the SARS-CoV-2 pathogenesis.

One of the limitations of this study is that it used in vitro models of SARS-CoV-2 infections in cell lines with a low multiplicity of infection (MOI) of 0.1, which hindered the biological relevance of the study results.

In future studies, further investigation with more sophisticated in vivo models such as ferrets could be used for an investigation related to full-length isoforms, differential polyadenylation, and transcript usage. The ideal MOI should also be determined through optimization studies.

Similarly, knock-down experiments with the same cell lines may be used to evaluate the effects of differentially expressed genes identified in this study. Furthermore, polyadenylation may be studied using cell lines with plasmids containing gene sequences of interest, followed by a poly(A) sequencing of varying lengths.

Further investigation is also needed to understand the link between increased polyadenylation of transcripts related to viral transcription in host cells after SARS-CoV-2 infection.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources