Jean and Peter Medawar wrote in 1977 that a virus is “simply a piece of bad news wrapped up in proteins.” The “bad news” in the SARS-CoV-2 case is the genome made of a very long ribonucleic acid (RNA) molecule. Grappling with COVID-19 pandemic, the world seems to be lost with no sense of direction in uncovering what this coronavirus (SARS-Cov-2) is composed of. Being an RNA virus, SARS-Cov-2 enters host cells and replicates its genomic RNA and produces many smaller RNAs (called “subgenomic RNAs”). These subgenomic RNAs are used for the synthesis of various proteins (spikes, envelopes, etc.) that are required for the beginning of SARS-Cov-2 lineage. Thus, the smaller RNAs make good targets for messing up new coronavirus’s conquering of our immune system. Though recent studies reported the sequence of the RNA genome, they only predicted where their genes might be, without concretely pinpointing what kind of genes are present and where exactly in the genome.

▲ Figure 1 The life cycle of SARS-CoV-2 When the spike protein of SARS-CoV-2 binds to the receptor of the host cell, the virus enters the cell, and then the envelope is peeled off, which let genomic RNA be present in the cytoplasm. The ORF1a and ORF1b RNAs are made by genomic RNA, and then translated into pp1a and pp1ab proteins, respectively. Protein pp1a and ppa1b are cleaved by protease to make a total of 16 nonstructural proteins. Some nonstructural proteins form a replication/transcription complex (RNA-dependent RNA polymerase, RdRp), which use the (+) strand genomic RNA as a template. The (+) strand genomic RNA produced through the replication process becomes the genome of the new virus particle. Subgenomic RNAs produced through the transcription are translated into structural proteins (S: spike protein, E: envelope protein, M: membrane protein, and N: nucleocapsid protein) which form a viral particle. Spike, envelope and membrane proteins enter the endoplasmic reticulum, and the nucleocapsid protein is combined with the (+) strand genomic RNA to become a nucleoprotein complex. They merge into the complete virus particle in the endoplasmic reticulum-Golgi apparatus compartment, and are excreted to extracellular region through the Golgi apparatus and the vesicle.

Led by Professors KIM Narry and CHANG Hyeshik, the Center for RNA Research within the Institute for Basic Science (IBS), South Korea, succeeded in dissecting the architecture of SARS-CoV-2 RNA genome, in collaboration with Korea Centers for Disease Control & Prevention (KCDC). The researchers experimentally confirmed the predicted subgenomic RNAs that are in turn translated into viral proteins. Furthermore, they analyzed the sequence information of each RNA and revealed where genes are exactly located on a genomic RNA. “Not only detailing the gene structure of SARS-CoV-2, we also discovered numerous new RNAs. Our work provides a high-resolution map of SARS-CoV-2. This map will help understand how the virus replicates and how it escapes the human defense system,” explains Professor KIM Narry, one of the corresponding authors of the study.

It was previously predicted that 10 subgenomic RNAs make up the viral particle structure. However, the research team confirmed that 9 subgenomic RNAs actually exist, invalidating one. Researchers also found that there are dozens of unknown subgenomic RNAs, owing to RNA fusion and deletion events. “Though it requires further investigation, these molecular events may lead to the relatively rapid evolution of coronavirus. It is unclear yet what these novel RNAs do, but a possibility is that they may assist the virus to avoid the attack from the host,” says Prof. Kim.

▲ Figure 2 Composition of genomic and subgenomic RNAs of SARS-CoV-2, and schematic diagram of virus particle structure

They believe if they figure out the unknown characteristics of RNA, the findings may offer a new clue for combatting the new coronavirus. Newly discovered features will also help to understand the life cycle of the virus and develop new strategies for antiviral therapy.

Behind the success of the study is the research team’s pairing of two complementary sequencing techniques; DNA nanoball sequencing and nanopore direct RNA sequencing. The nanopore direct RNA sequencing allows to directly analyze the entire long viral RNA without fragmentation. Conventional RNA sequencing methods usually require a step-by-step process of cutting and converting RNA to DNA before reading RNA. Meanwhile, the DNA nanoball sequencing can read only short fragments, but has the advantage of analyzing a large number of sequences with high accuracy. These two techniques turned out to be highly complementary to each other to analyze the viral RNAs.

“Now we have secured a high resolution gene map of the new coronavirus that guides us where to find each bit of genes on all of the total SARS-CoV-2 RNAs (transcriptome) and all modifications RNAs (epitranscriptome). It is time to explore the functions of the newly discovered genes and the mechanism underlying viral gene fusion. We believe that our study will contribute to the development of diagnostics and therapeutics to combat the virus more effectively,” notes Professor KIM Narry.

Dahee Carol Kim
IBS Communications Team