In a study recently published in the journal Nature Communications, scientists have mapped the interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and host RNA. They have observed that the virus heavily methylates its genome using the methylation machinery of the host RNA, leading to an improvement in viral fitness.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pathogen of coronavirus disease 2019 (COVID-19), is an enveloped, positive-sense, single-stranded RNA virus with a genome size of around 30 kb. The viral genome encodes four structural proteins, including the envelope, membrane, nucleocapsid, and spike proteins, which are required for viral budding and host cell entry processes.
In addition, the genome encodes multiple accessory proteins that are vital for maintaining the viral lifecycle inside host cells. Specifically, accessory protein-mediated viral RNA replication leads to the generation of a full-length viral genome and multiple subgenomic RNAs (sgRNAs). The full-length viral RNA and sgRNAs interact with host cell proteins and RNAs to regulate viral propagation inside infected cells.
Since its emergence in China in December 2019, SARS-CoV-2 has acquired more than 12,000 mutations, leading to the emergence of multiple viral variants. Although spike mutations are predominant in the SARS-CoV-2 genome, some deletion mutations in the ORF8 region have been identified in many countries, including Singapore, Australia, Bangladesh, Taiwan, and Spain. Particularly in Singapore, a 382-nucleotide deletion (Δ382) has been found in the viral genome, which causes truncation of ORF7 and deletion of ORF8. Compared to wildtype SARS-CoV-2, variants containing Δ382 induce relatively mild infections in infected patients.
In the current study, the scientists have investigated RNA-RNA interactions between the wildtype SARS-CoV-2 and Δ382 mutant inside host cells. In addition, they have examined the host-virus interactions to identify functional elements across the viral genome.
Specifically, they have utilized various high-throughput RNA techniques to examine secondary structures within the viral genome. In addition, they have conducted proximity ligation sequencing to identify host RNA – viral RNA interactions inside infected cells.
Structural arrangements of the viral genome
The study findings indicated that both wildtype virus and Δ382 mutant maintain highly stable and consistent genomic structure with limited alternative folding inside host cells. Twelve functional, structural elements were identified within the viral genome. In addition, a total of 21 single-stranded regions were identified that could be potentially used for COVID-19 treatment using siRNA targeting approaches.
Regarding pair-wise interactions across the viral genome, the study identified 237 and 187 intramolecular interactions in the wildtype virus and Δ382 mutant, respectively. The majority of these interactions were transiently-formed long-range interactions (>1 kb). With further analysis, it was observed that ribosome pause sites contain more pair-wise interactions, indicating that RNA structures play a vital role in regulating the translation of the viral genome.
By comparing the genomes of wildtype virus and Δ382 mutant, it was observed that these pair-wise interactions are differentially arranged in two viruses. In addition, structural differences in genomic RNA and sgRNA were observed between the wildtype virus and Δ382 mutant.
The long-read sequencing analysis conducted in the study revealed that sgRNAs have different structures from the full-length genomic RNA and that different sgRNAs could gain different structural arrangements despite sharing the same sequences. Among various sgRNAs, the ORF7b sgRNA showed the highest single-strandedness in both wildtype virus and Δ382 mutant.
Host – virus interactions
A total of 374 and 334 host RNAs were identified that interacted with the genomes of wildtype virus and Δ382 mutant, respectively. The highest interactions were observed between the viral RNA and host mitochondrial and small nuclear RNAs. Upon SARS-CoV-2 infection, a preferential translation and stabilization of strong interactors were observed.
Among identified small nuclear RNAs, SNORD27 showed the strongest interaction with viral RNA. This RNA is known to regulate 2’-O-methylation of 18 S ribosomal RNA. As observed in the study, the interaction between SNORD27 and viral RNA resulted in extensive 2’-O-methylation of the viral genome, which was 19-fold higher than the modifications observed in host mRNAs.
Host RNAs that interacted with the viral RNA exhibited higher methylation, whereas no modification was observed at sites that were located far way. This indicates that the virus sequesters methylation machinery from host RNAs towards its genome. Because of a generalized loss of 2’-O-methylation on host RNA, a reduction in cellular RNA was observed upon viral infection.
The study highlights an important observation that SARS-CoV-2 captures RNA methylation enzymes of host cells to destabilize host RNA and to reduce its abundance. These changes in host cells subsequently facilitate viral replication and improve viral fitness.