In a recent study published on 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.
Study: Viral and host small RNA transcriptome analysis of human cells infected with SARS-CoV-1 and SARS-CoV-2 reveals novel viral short RNAs. Image Credit: CROCODILE/Shutterstock
The coronavirus disease 2019 (COVID-19) pandemic resulted in widespread mortality and morbidity around the world. Although its diagnosis and treatment required unexpected resources, it is still important to have a better understanding 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 investigation. The comparative stability of small RNAs could prove useful in the development of diagnostic and prognostic tools for the detection of SARS-CoV-2 in samples for timely diagnosis.
About the study
In the present study, the researchers described small perturbations in RNA expression 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 infected with SARS-CoV-2 or SARS-CoV-1. The small RNA sequencing reads observed in the mock, as well as in the infected cells, were first excised from the adapters before being selected for size and aligned to the related viral reference genome. The team also calculated the counts per million (CPM) values associated with the assigned reads 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 with the SARS-CoV-2 or SARS-CoV-1 genomes after removing overlapping fragments. In addition, 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 tested three negative control primers that fused to regions within the SARS-CoV-2 genome. In addition, analysis of differential expressions was performed for control cells with mock infection versus cells infected with SARS-CoV-2 and SARS-CoV-2 24 hours post infection.
Results of the study showed that infection with SARS-CoV-2 or SARS-CoV-1 resulted in significant proliferation of short RNA fragments that were derived from forward and reverse viral RNA genomic strands four, 12, and 24 hours later. of the infection. of Calu-3 cells. In addition, the number of reads that mapped to forward viral RNA strands was nearly 20-fold higher than those that aligned to reverse strands. This could be the result of increased protection of the positive strands of the genome by ribosomes or other related proteins.
The team also observed that the viral sequences, which perfectly matched the small RNA fractions of the infected Calu-3 cells, represented 2.83% of the total infected with SARS-CoV-1 and 1.51% of the total. of SARS-CoV-2. -Infected cells. This approximately two-fold distinction could be due to the markedly higher cytotoxicity associated with SARS-CoV-2.
The team also noted that several reads aligned to individual positions in the SARS-CoV-2 or SARS-CoV-1 genomes. In addition, five putative forward-stranded svRNAs showed significantly higher signal from infected cells compared to background. Furthermore, the influence of the addition of poly(A) tails to the small RNAs suggested that the detected vsRNAs had amplification from small RNA templates rather than full-length viral RNA. For two of the forward-stranded svRNAs, a similar signal was observed for infected and uninfected cells. In addition, the third negative control was found to be induced within the infected cells. However, the team noted that the amplification was found later than in the putative SARS-CoV-2 small RNA that had the most recent amplification. This suggested that the SARS-CoV-2 genome could encode small RNA fragments with a discrete size.
Differential expression analysis showed that a significantly higher number of small RNA transcripts showed differential expression in SARS-CoV-2 compared to SARS-CoV-1 infection. Notably, among the 14,747 transcripts remaining after low expression filtering, zero downregulated and 12 upregulated sequences were detected in SARS-CoV-1 infection. On the other hand, 120 downregulated and 268 upregulated sequences were detected in SARS-CoV-2 infection.
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 observed that SARS-CoV-2 caused differential expression of a large number of small RNAs compared to SARS-CoV-1 infection. Furthermore, the small RNA fragments were mainly aligned with IncRNA and protein-coding genes. Furthermore, although substantial alterations in small RNA expression were observed, some micro RNAs (miRNAs) were affected, suggesting the limited role 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 diagnosis of COVID-19 or identification of persistent infections.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice or health-related behavior, or treated as established information.