5' UTR barcode of the 2019 novel Coronavirus leads to insights into its virulence

"Novel coronavirus 2019" (which was renamed subsequently "severe acute respiratory syndrome coronavirus-2" (SARS-CoV-2) on 11 February 2020) caused a pneumonia outbreak in Wuhan (Hubei Province, China) in December 2019. In our previous studies, two important findings regarding SARS-CoV-2 were reported, for the first time, on 21 January 2020: (1) multiple alternative translations of a coding sequence in genomes of betacoronavirus subgroup B;(2) a novel mutation in the spike (S) proteins of betacoronavirus. By this mutation, SARS-CoV-2 acquired a cleavage site for the furin enzyme in its S protein, which is not present in the S proteins of most other betacoronaviruses (e.g. SARS-CoV). In the present study, we performed analyses of 5' untranslated regions (UTRs) in betacoronavirus. Using 5' UTR barcodes, 1,265 betacoronaviruses were clustered into four classes, and viruses in each class had similar virulence. The class 1, 2, 3 and 4 match the subgroup C, B, A and D of betacoronavirus, respectively. In particular, SARS-CoV-2 and SARS-CoV have the same 5' UTR barcode. As the main contribution of the present study, we developed 5' UTR barcoding to be used in the detection, identification, classification and phylogenetic analysis of, but not limited to coronavirus. Our method is very useful for early-warning, prevention and control of coronavirus. We found that Internal Ribosome Entry Sites (IRESs) may have important roles in the virulence of betacoronavirus. This important finding is reported, for the first time, to understand the virulence of SARS-CoV-2 at the molecular level. This finding can be used directly for vaccine development and design of drugs against SARS-CoV-2, but such development is not limited to coronavirus only. In addition, we propose that the upstream hairpin structures neighboring the start codons in mRNAs have important roles in protein translation in eukaryotes.

How the Replication and Transcription Complex Functions in Jumping Transcription of SARS-CoV-2.

Background: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although unprecedented efforts are underway to develop therapeutic strategies against this disease, scientists have acquired only a little knowledge regarding the structures and functions of the CoV replication and transcription complex (RTC). Ascertaining all the RTC components and the arrangement of them is an indispensably step for the eventual determination of its global structure, leading to completely understanding all of its functions at the molecular level. Results: The main results include: 1) hairpins containing the canonical and non-canonical NSP15 cleavage motifs are canonical and non-canonical transcription regulatory sequence (TRS) hairpins; 2) TRS hairpins can be used to identify recombination regions in CoV genomes; 3) RNA methylation participates in the determination of the local RNA structures in CoVs by affecting the formation of base pairing; and 4) The eventual determination of the CoV RTC global structure needs to consider METTL3 in the experimental design. Conclusions: In the present study, we proposed the theoretical arrangement of NSP12-15 and METTL3 in the global RTC structure and constructed a model to answer how the RTC functions in the jumping transcription of CoVs. As the most important finding, TRS hairpins were reported for the first time to interpret NSP15 cleavage, RNA methylation of CoVs and their association at the molecular level. Our findings enrich fundamental knowledge in the field of gene expression and its regulation, providing a crucial basis for future studies.

A furin cleavage site was discovered in the S protein of the 2019 novel coronavirus

The 2019 novel Coronavirus (2019-nCoV) has caused the pneumonia outbreak in Wuhan (a city of China). In our previous study, the analytical results showed that both 2019-nCoV and SARS coronavirus belong to Betacoronavirus subgroup B (BB coronavirus), but have large differences, which are consistent with the differences in the clinical symptoms of two related diseases. The most important finding was that the alternative translation of Nankai CDS could produce more than 17 putative proteins, which may be responsible for the host adaption. The genotyping of 13 viruses using the 17 putative proteins revealed the high mutation rate and diversity of BB coronavirus. The present study for the first time (on January 21st, 2020) reported a very important mutation in the Spike (S) proteins of Betacoronavirus. By this mutation, 2019-nCoV acquired a cleavage site for furin enzyme in its S protein, which is not present in the S proteins of most other Betacoronavirus (e.g. SARS coronavirus). This cleavage site may increase the efficiency of virus infection into cells, making 2019-nCoV has significantly stronger transmissibility than SARS coronavirus. The infection mechanism of 2019-nCoV may be changed to being more similar to those of MHV, HIV, Ebola virus (EBoV) and some avian influenza viruses, other than those of most other Betacoronavirus (e. g. SARS coronavirus). In addition, we unexpectedly found that some avian influenza viruses acquired a cleavage site for furin enzyme by the similar mutation as 2019-nCoV. Therefore, the natural mutation can result in a short insertion to form a cleavage site for furin enzyme. The cleavage site for furin enzyme in 2019-nCoV contains the "CGGCGG" sequence encoding two arginine (R) residues. "CGG", however, is a rare codon for human. So we concluded that these two codons were present in the 2019-nCoV -like Betacoronavirus before they transmitted into human and the intermediate host (s) are mammals with a high relative frequency of "CGG" usage. We provide a relative frequency table of " CGG" usage in mammals to help identify the intermediate hosts of 2019-nCoV. Future studies of this mutation will help to reveal the stronger transmissibility of 2019-nCoV and lay foundations for vaccine development and drug design of, but not limited to 2019-nCoV.

Bioinformatics analysis of the 2019 novel coronavirus genome

In December 2019, a pneumonia outbreak caused by a human coronavirus was reported in Wuhan (China). This virus was predicted as a new coronavirus, named the 2019 novel coronavirus (2019-nCoV), as it caused clinical symptoms different from Severe Acute Respiratory Syndrome (SARS) during the 2003 outbreak. Currently, most of the researchers simply use the complete genome or specific structural gene sequences to investigate coronavirus (e. g. phylogenetic analysis) without considering the functions of the products from coronavirus genes. To overcome this shortcoming, we proposed the joint analysis of the molecular function and phylogeny, and applied it in our previous study of genomes of Betacoronavirus subgroup B(BB coronavirus). In that study, we identified a 22-bp complemented palindrome from a highly conserved Coding Sequence (CDS). Both the 22-bp complemented palindrome (named Nankai complemented palindrome) and the CDS (named Nankai CDS), evolutionary conserved in BB coronavirus genomes, were identified as genomic features associated to the molecular functions of BB coronavirus. In the present study, we used these two genomic features to trace the origin of 2019-nCoV (GenBank: MN908947) and conduct a preliminary study of the mechanisms in the cross-species infection and host adaption of BB coronavirus. Our analytical results show that 2019-nCoV with large differences from the SARS coronavirus, may originate from BB coronaviruses in bats. The most important finding is that the alternative translation of Nankai CDS could produce more than 17 putative proteins, which may be responsible for the host adaption. The genotyping of 13 viruses using the 17 putative proteins revealed the high mutation rate and diversity of BB coronavirus. Our study, for the first time, aimed to explain the reason for the high host adaptability of the multi-host BB coronavirus at the molecular level using large amounts of genomic data. The findings in the present study laid foundations for the rapid detection, genotyping, vaccine development and drug design of, but not limited to BB coronavirus.