Viral short RNAs detected in SARS-CoV-2 RNA transcriptome

By Bhavana KunkalikarReviewed by Aimee MolineuxJan 3 2023

In a recent study posted to the bioRxiv* preprint server, researchers analyzed the ribonucleic acid (RNA) transcriptome associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1)-infected human cells.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

The coronavirus disease 2019 (COVID-19) pandemic resulted in widespread mortality and morbidity across the globe. While its diagnosis and treatment needed unexpected resources, it is still important to have better knowledge of the molecular perturbations associated with COVID-19. Several studies of the SARS-CoV-2 transcriptome involve long RNA biotypes, while small RNAs need further research. The comparative stability of small RNAs could prove useful in developing prognostic and diagnostic tools for SARS-CoV-2 detection in samples for timely diagnosis.  

About the study

In the present study, researchers described small RNA expression perturbations observed after SARS-CoV-2 and SARS-CoV-1 infection.

To assess the characteristics of short viral RNAs (svRNAs), the team studied small RNA-sequencing datasets derived from Calu-3 cells that were either SARS-CoV-2 or SARS-CoV-1-infected. Small RNA-sequencing reads observed in the mock as well as infected cells, were first cut from adapters before they were selected according to size and aligned as per the related viral reference genome. The team also calculated the count-per-million (CPM) values associated with the reads mapped for each reference.

The team also estimated the distribution pattern associated with viral short RNA (vsRNA) reads and detected virus-specific fragments mapped to different locations. This was achieved by extracting reads that aligned to either SARS-CoV-2 or SARS-CoV-1 genomes after eliminating overlapping fragments. Furthermore, RNA from uninfected and uninfected A549 ACE-2 cells was used to analyze the detection of five selected small RNAs. Reverse transcription and polyadenylation were performed before real-time quantitative polymerase chain reaction (qPCR). The team also assessed three negative control primers that were annealed to regions within the SARS-CoV-2 genome. Additionally, differential expressions analysis was conducted for mock-infected control cells versus SARS-CoV-2 and SARS-CoV-2-infected cells 24 hours after infection.      

Results

The study results showed that infection with SARS-CoV-2 or SARS-CoV-1 resulted in the significant proliferation of short RNA fragments which were obtained from reverse as well as forward viral RNA genome strands four, 12, and 24 hours post-infection of the Calu-3 cells. Also, the number of reads that were mapped as per the forward viral RNA strands was almost 20 times more than those that were aligned as per the reverse strands. This could be a result of higher protection of positive genome strands by either ribosomes or other related proteins.

The team also noted that the viral sequences, which were a perfect match within the small RNA fractions of infected Calu-3 cells, were 2.83% of the total SARS-CoV-1-infected and 1.51% of the total SARS-CoV-2-infected cells. This approximately two-fold distinction could be because of the remarkably higher cytotoxicity associated with SARS-CoV-2.

The team also noted that several reads aligned to single positions in the SARS-CoV-2 or SARS-CoV-1 genomes. Additionally, five putative forward-strand svRNAs showed a significantly increased signal of infected cells in comparison to the background. Furthermore, the influence of the poly(A) tails addition to the small RNAs suggested that the detected vsRNAs had amplification from small RNA templates instead of full-length viral RNAs. For two of the forward-strand svRNAs, a similar signal was observed for the uninfected and infected cells. Moreover, the third negative control was found to be induced within the infected cells. Yet, the team noted that amplification was found later than that in the putative SARS-CoV-2 small RNA having the most recent amplification. This suggested that the SARS-CoV-2 genome could encode small RNA fragments having a discrete size.       

Differential expression analysis showed that a significantly higher number of small RNA transcripts displayed differential expression in SARS-CoV-2 as compared to that in SARS-CoV-1 infection. In particular, among the 14,747 transcripts that remained post-low-expression filtering, zero down- and 12 up-regulated sequences were detected in the SARS-CoV-1 infection. On the other hand, 120 down- and 268 up-regulated sequences were detected in the SARS-CoV-2 infection.

Conclusion

The study findings highlighted significantly different reactions of small RNA expressions when Calu-3 cells were infected with SARS-CoV-2 and SARS-CoV-1. Thus, it was noted that SARS-CoV-2 elicited differential expression of a huge number of small RNAs in comparison to that in SARS-CoV-1 infection. Furthermore, the small RNA fragments aligned primarily to IncRNA and protein-coding genes. Additionally, while substantial alterations were observed in small RNA expression, a few micro RNAs (miRNAs) were impacted, which suggested the limited function of host miRNAs in infection progression.

The researchers believe that extensive research is required to assess the mechanisms involved in the biological function as well as the biogenesis of svRNAs. The team also noted that it is possible that svRNAs derived from SARS-CoV-2 could serve as novel ultrasensitive biomarkers for early COVID-19 diagnosis or identification of persistent infection.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.