Coronavirus genomes are single-stranded mRNAs, containing both coding and untranslated regions (UTRs). The 5′UTR and 3′UTR are crucial for coronavirus RNA replication, transcription, and dominating host systems biology [1
]. However, their exact roles and their evolutions, specifically in the new SARS-CoV-2, are mainly unknown.
There are some 5′UTR regions that are vital for virus replication (Figure 1
). A specific region of 70 nucleotides at the 5′ end of the genome, referred as the ‘leader’ sequence, has been found at the 5′ ends of all encoded transcripts, highlighting its importance. A conserved cis-acting element, named transcription regulatory sequence (TRS), immediately follows the leader sequence, representing a unique feature of coronaviruses.
Compared to the other positive-strand RNA viruses, coronaviruses have a unique and complicated pattern of continuous and discontinuous RNA synthesis (Figure 2
). Like the other positive-strand RNA viruses, continuous RNA synthesis happens for genome replication to yield multiple copies of the genomic RNA. In continuous RNA synthesis, a full-length complementary negative-strand RNA is utilized as the template for generation of progeny virus genomes [2
]. In discontinuous transcription, during the synthesis of sub-genomic negative-strand RNAs, a premature termination and template switch occur to add copies of the leader sequence [2
]. The presence of the 5′ leader sequence provides an efficient strategy for the efficient accumulation of coronaviruses mRNAs and proteins during infection because of the protection of viral mRNAs from endonucleolytic cleavage of the capped mRNAs [3
]. Furthermore, the complement of the leader sequence supports initiation of positive-strand RNA synthesis, generating negative-strand sub-genomic RNAs as templates for further productions of positive-strand sub-genomic mRNAs [2
Whereas UTR is a non-coding region, another unique structure of coronaviruses is the existence of a short open reading frame (ORF) on one of the 5′UTR stem loops. In bovine coronavirus, maintenance of the short ORF is positively correlated with viral RNA accumulation [4
]. The other non-coding region, 3′-UTR folds into a unique stem-loop two motif secondary structure that is required for virus viability.
UTRs are also important in the context of host microRNA interaction. MicroRNAs play their negative regulatory roles via sequence-specific interactions with the 3′ or 5′UTRs [5
]. Consequently, sequence variation in the viral UTR regions can prevent the binding of human microRNAs, so preventing microRNA-based immunity [6
]. The problem is more serious in case of SARS-CoV-2 which originated recently from bat, such that human microRNAs have not evolved to interact with their UTRs.
Here, we hypothesize that the TRS and leader sequences of 5′ UTRs are crucial for coronavirus RNA replication. Moreover, microRNAs can bind to the TRS and leader sequences of the 5′UTR to reduce SARS-CoV-2 replication. The first aim of this study was to unravel the evolution of the TRS and leader sequences in 5′UTR regions of SARS-CoV-2. The second aim was to identify inhibitory microRNAs, present in human and other organisms, that can bind to the key regulatory regions of SARS-CoV-2 sequences.
UTRs, particularly in the 5′UTR, are of high translational importance. The leader sequence and TRS in the 5′ non-coding part of SARS-CoV-2 can be considered as the Achilles’ heel of SARS-CoV-2. Leader sequence is located at the 5′ ends of all encoded transcripts, highlighting its potential significance. The TRS can explain the host range and pathogenicity of a coronavirus. UTRs are potential sites for antiviral drugs to bind and inhibit the virus replication. There is no report on disruption of 5′UTR in SARS-CoV-2 but, in bovine coronavirus, it has been found that disruption of either stem-loop III or stem-loop IV of the 5′UTR stops virus RNA replication, suggesting that these regions function as cis-acting elements [4
]. On the other hand, microRNAs that bind to SARS-CoV-2 UTRs can be induced by drugs or food supplements to reduce virus replication. Enhancing host microRNA defense machinery against the 5′UTR region of a virus can help in prevention of SARS-CoV-2 infection. The above-mentioned strategies represent potentially achievable treatments against COVID-19 infection. In this study, we presented a model of literature mining-based drug discovery to induce inhibitory microRNAs against leader sequence of SARS-CoV-2.
Some host cell microRNAs are important components of the host immune defense against viral infection as they destroy the viral RNA [21
]. In this study, we found that hsa-miR-5004-3p is a unique human microRNA with the ability to target the leader sequence of SARS and SARS-CoV-2. Our comprehensive meta-analysis also documented a significant (p
< 0.01) decline in the expression of MIR-5004-3p after SARS-COV-2 infection in trachea, lung, and bronchial organoids as well as in lung-derived Calu-3 and A549 cells. Interestingly, a decreased level of hsa-miR-5004-3p has been reported in the whole blood of patients during dengue virus (DENV) infection. hsa-miR-5004-3p was undetectable in early DENV infection, but expression was high in most of the healthy controls and recovered dengue patients. This finding demonstrated suppression of hsa-miR-5004-3p during the early phases of DENV infection and its importance in patient recovery [22
]. Like COVID-19, DENV is a rampant arboviral illness worldwide [22
] with high viral transmission success and immune evasion.
We found a significant trend in the 5′UTR of SARS-CoV-2 to escape from binding of hsa-miR-5004-3p by insertion-type mutation. Such mutations decrease microRNA-5′UTR binding stability and allow SARS-CoV-2 to escape the available human microRNA immunity system. We suggest that the lack of innate human inhibitory microRNAs for SARS-CoV-2 contributes to its high replication in the infected human cells. On the other hand, mining of two hundred million deposited human genomic variants led us to discover splice-disrupted mutations in the genomic structure of hsa-miR-5004-3p. These mutations can negatively affect hsa-miR-5004-3p function in preventing SARS-CoV-2 replication.
The abundance of some human inhibitory microRNAs against SARS-CoV-2 is associated with age [19
]. It has been discussed that higher COVID-19 virulence in the aged patients [23
] is potentially related to the decline in microRNAs in elderly people [19
]. In addition to age, some of the human microRNAs downregulate in coronary artery disease, kidney disease, colorectal cancer, osteosarcoma, prostate cancer, obesity/diabetes, myocardial injury, hepatocellular carcinoma, non-small cell lung cancer, gastrointestinal tumors, and colorectal cancer [19
]. Like age, the mentioned conditions can also contribute to lower abundance of human inhibitory microRNAs and consequently, higher severity and mortality of COVID-19.
This hypothesis needs to be examined in future studies.
The 5′UTR inhibitory microRNAs, such as hsa-miR-5004-3p or those of plant origin such as ptc-miR474 and csi-miR3953, have promising potential for developing new therapies. MicroRNA vaccine, nanoparticles, synthesized microRNAs, microRNA exosome, and dietary microRNAs can be considered in this context. MicroRNA-peptide fusion is also suggested as potential vaccine candidates [24
]. In addition to hsa-miR-5004-3p, plant microRNAs with very low binding energy (less than −22 kcal/mol) against leader sequence of 5′UTR of SARS-CoV-2, such as ptc-miR474b and ptc-miR474a (Populus trichocarpa
) and csi-miR3953 (Citrus sinensis
) are possible candidates for inclusion. Nanoformulations of the SARS-CoV-2 inhibitory microRNAs can successfully and safely deliver microRNAs to lung cells, to reduce viral replication in the host cell and suppress the viral load [25
]. MicroRNA-based nanoparticles can also be utilized in the form of nanovaccines to prevent SARS-CoV-2 infection. Compared with traditional vaccines, nanovaccines have many potential benefits, including: (1) Specific targeting to infection sites, (2) minimal off-target effects, and (3) the possibility of nasal spray/drop formulation for direct activation of the immune system in the respiratory tract [25
]. Due to shortness of microRNA sequences (18–25 nucleotides), synthesizing of inhibitory microRNAs are readily conducted. It has been suggested that mixtures of synthesized microRNAs, called multiple microRNA cocktail, targeting different coding (such as S gene) and UTR regions of SARS-CoV-2 can be considered as new COVID-19 treatment strategy [19
]. This synthesized mixture may be delivered to the target host cells by liposomes like exosome (extracellular vesicle), polymer-based carriers, or nanoparticles [26
]. High potential of microRNA exosomes as biomarkers of infection and recovery in COVID 19 has been suggested [24
]. On the other hand, the combination of a mRNA vaccine with the immune promoting agents, such as inhibitory microRNAs, can increase its efficiency and protection, even in the case of new SARS-CoV-2 mutations.
Interestingly, expression of microRNAs, complementary to the 3′UTR of viruses, is the main protecting strategy in plants that degrades the viral RNA or blocks its translation by ribosomes [26
]. Plants are a rich source for microRNAs, with remarkable therapeutic and preventive roles in many diseases [27
]. Recently, it has been suggested that microRNAs in food can be absorbed by the human circulatory system [28
]. The absorbed microRNA, named dietary microRNA, can regulate gene expression and biological processes in mammalian cells [28
]. Dietary microRNA, as a novel functional feature of food [28
], opens a new potential avenue for safe and accessible COVID19 protection and treatment. Further research on plant dietary microRNAs may lead to a safe treatment strategy against COVID19 by oral use of plant microRNAs or whole plants containing SARS-CoV-2 inhibitory microRNAs.
Sequence variation in the non-coding viral genomic regions may predispose people to develop more severe disease. It should be noted that the genetic signature of the pathogenesis severity in the non-coding regions of SARS-CoV-2 is still unknown. A recent report on human–mink–human transmission cycle [29
] highlights the necessity to uncover all functional mutations, including the ones that occur in UTR regions. The availability of 212,346 (at 21 November 2020) SARS-CoV-2 genomic sequences, including 159,057 sequences of the full genome and high coverage, in GISAID (https://www.gisaid.org/
] and NCBI provides the chance of pattern recognition in 5′UTR sequences of SARS-CoV-2, particularly against host microRNA inhibitory machinery, by machine learning models. Models and statistics such as decision tree classification based on association rule mining and deep learning [31
] that have been used for eukaryotic promoter and UTR analysis, can be examined for UTR analysis of SARS-CoV-2. It should be noted that some of the sequences that have been deposited in GISAID as full genomes have incomplete or low-quality sequencing in 3′UTR and 5′UTR regions. Data cleaning of 5′UTR and 3′UTR regions is a major task before analysis as many of the announced SARS-CoV-2 full genomes with complete coding sequences have incomplete, short, or low-quality UTR sequences. This problem casts doubt on the reliability of the currently identified SARS-CoV-2 sequences and UTR mutations.
Non-coding regions are crucial for SARS-CoV-2 replication, transcription, and domination of host systems biology. Unravelling the reasons underpinning SARS-CoV-2 success in dominating human cells and its high transmission rate is crucial for future research. The present study is a significant step towards unravelling the evolution of the 5′UTR in SARS-CoV-2, discovering the key regions, and utilizing the UTRs for lowering virus load in the infected cells. We have shown that human hsa-miR-5004-3p and several plant microRNAs are good microRNA candidates to target the leader sequence of SARS-CoV-2 at 5′UTR region.
In this study, an evolutionary pattern in the 5′UTR of SARS-CoV-2 from SARS was discovered whereby SARS-CoV-2 tries to escape hsa-miR-5004-3p binding by the generation of insertion-type mutations. The lack of a human inhibitory microRNAs to target the 5′ UTR of SARS-CoV-2 can contribute in immunity evasion and pathogenesis in SARS-CoV-2. Decline in expression of human inhibitory microRNAs with increasing age, and in a range of diseases such as diabetes and obesity, can also contribute to higher mortality of SARS-CoV-2 in elderly patients and in individuals suffering from these diseases.
We also developed a literature mining-based drug repurposing strategy to induce inhibitory microRNAs that are potentially active against the leader sequence of SARS-CoV-2. Activation of inhibitory machinery microRNAs by drug repurposing and food supplements are easily achievable treatment strategies against COVID-19. Additionally, the microRNAs identified can be utilized for other therapeutic strategies such as development of microRNA vaccines, nanoparticles, synthesized microRNAs, microRNA exosome, and dietary microRNA.