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.

Genomic Feature Analysis of Betacoronavirus Provides Insights Into SARS and COVID-19 Pandemics.

In December 2019, the world awoke to a new betacoronavirus strain named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Betacoronavirus consists of A, B, C and D subgroups. Both SARS-CoV and SARS-CoV-2 belong to betacoronavirus subgroup B. In the present study, we divided betacoronavirus subgroup B into the SARS1 and SARS2 classes by six key insertions and deletions (InDels) in betacoronavirus genomes, and identified a recently detected betacoronavirus strains RmYN02 as a recombinant strain across the SARS1 and SARS2 classes, which has potential to generate a new strain with similar risk as SARS-CoV and SARS-CoV-2. By analyzing genomic features of betacoronavirus, we concluded: (1) the jumping transcription and recombination of CoVs share the same molecular mechanism, which inevitably causes CoV outbreaks; (2) recombination, receptor binding abilities, junction furin cleavage sites (FCSs), first hairpins and ORF8s are main factors contributing to extraordinary transmission, virulence and host adaptability of betacoronavirus; and (3) the strong recombination ability of CoVs integrated other main factors to generate multiple recombinant strains, two of which evolved into SARS-CoV and SARS-CoV-2, resulting in the SARS and COVID-19 pandemics. As the most important genomic features of SARS-CoV and SARS-CoV-2, an enhanced ORF8 and a novel junction FCS, respectively, are indispensable clues for future studies of their origin and evolution. The WIV1 strain without the enhanced ORF8 and the RaTG13 strain without the junction FCS "RRAR" may contribute to, but are not the immediate ancestors of SARS-CoV and SARS-CoV-2, respectively.

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.