SARS-CoV-2 Alters Human Host RNA To Improve Viral Fitness


Study findings by researchers from the Agency for Science, Technology and Research-Singapore ( (A*STAR), Duke-NUS Medical School-Singapore and the Yong Loo Lin School of Medicine at the National University of Singapore have alarmingly found that the SARS-CoV-2 coronavirus is able to destabilize and also alter human host RNA in order to for it to improve its own viral fitness.

The study team investigated the RNA structure and RNA-RNA interactions of wildtype (WT) and a mutant (Δ382) SARS-CoV-2 in cells using Illumina and Nanopore platforms.
The team identified twelve potentially functional structural elements within the SARS-CoV-2 genome and observed that subgenomic RNAs can form different structures, and that WT and Δ382 virus genomes fold differently.
By utilizing proximity ligation sequencing, the study team identified hundreds of RNA-RNA interactions within the virus genome and between the virus and host RNAs.
The team found that the SARS-CoV-2 genome binds strongly to mitochondrial and small nucleolar RNAs and is extensively 2’-O-methylated.

2’-O-methylation sites are enriched in viral untranslated regions, associated with increased virus pair-wise interactions, and are decreased in host mRNAs upon virus infection, suggesting that the virus sequesters methylation machinery from host RNAs towards its genome.
The study findings were published in the peer reviewed journal: Nature Communications
The study team basically had mapped the interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and host RNA.

The team found that the novel coronavirus heavily methylates its genome using the methylation machinery of the host RNA, leading to an improvement in viral fitness. In the process the human host RNAs are destabilized and also altered.
The SARS-CoV-2 coronavirus, 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.
Importantly the virus 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.   
From its debut in Wuhan 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.
It was found that 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.
However despite mild infections, what damages it does to the human host in the long term and also the medical conditions that can arise over time have not been established yet nor studied in detail yet.

Destabilization and alterations of the RNAs of the human host are bound to result in long term medical conditions arising.
For the study, the researchers investigated RNA-RNA interactions between the wildtype SARS-CoV-2 and Δ382 mutant inside host cells. In addition, they examined the host-virus interactions to identify functional elements across the viral genome.
In particularly, they utilized various high-throughput RNA techniques to examine secondary structures within the viral genome. In addition, the study team had conducted proximity ligation sequencing to identify host RNA – viral RNA interactions inside infected cells.
The observed 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.
With regards to pair-wise interactions across the viral genome, the research team identified 237 and 187 intramolecular interactions in the wildtype virus and Δ382 mutant, respectively.
Importantly 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.
Through detailed analysis and 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.
Also 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.
Also important to take note of, a total of 374 and 334 host RNAs were identified that interacted with the genomes of wildtype virus and Δ382 mutant, respectively.
It was found that 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.
Significantly 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.
Human 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.
Alarmingly the study highlights the 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.
Further detailed studies are warranted urgently to know how ‘disruptions’ in these human host cells will affect long term health conditions.
The implications for Long COVID and the serious medical conditions that can arise in the long term are extremely worrying.

Continuous or recurrent positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR tests have been reported in samples taken from patients weeks or months after recovery from an initial infection (1–17). Although bona fide reinfection with SARS-CoV-2 after recovery has recently been reported (18), cohort-based studies with subjects held in strict quarantine after they recovered from COVID-19 suggested that at least some “re-positive” cases were not caused by reinfection (19, 20).

Furthermore, no replication-competent virus was isolated or spread from these PCR-positive patients (1–3, 5, 6, 12, 16), and the cause for the prolonged and recurrent production of viral RNA remains unknown. SARS-CoV-2 is a positive-stranded RNA virus. Like other beta-coronaviruses (SARS-CoV-1 and Middle East respiratory syndrome-related coronavirus), SARS-CoV-2 employs an RNA-dependent RNA polymerase to replicate its genomic RNA and transcribe subgenomic RNAs (21–24).

One possible explanation for the continued detection of SARS-CoV-2 viral RNA in the absence of virus reproduction is that, in some cases, DNA copies of viral subgenomic RNAs may integrate into the DNA of the host cell by a reverse transcription mechanism. Transcription of the integrated DNA copies could be responsible for positive PCR tests long after the initial infection was cleared.

Indeed, nonretroviral RNA virus sequences have been detected in the genomes of many vertebrate species (25, 26), with several integrations exhibiting signals consistent with the integration of DNA copies of viral mRNAs into the germline via ancient long interspersed nuclear element (LINE) retrotransposons (reviewed in ref. 27).

Furthermore, nonretroviral RNA viruses such as vesicular stomatitis virus or lymphocytic choriomeningitis virus (LCMV) can be reverse transcribed into DNA copies by an endogenous reverse transcriptase (RT), and DNA copies of the viral sequences have been shown to integrate into the DNA of host cells (28⇓–30). In addition, cellular RNAs, for example the human APP transcripts, have been shown to be reverse-transcribed by endogenous RT in neurons with the resultant APP fragments integrated into the genome and expressed (31).

Human LINE1 elements (∼17% of the human genome), a type of autonomous retrotransposons, which are able to retro-transpose themselves and other nonautonomous elements such as Alu, are a source of cellular endogenous RT (32–34). Endogenous LINE1 elements have been shown to be expressed in aged human tissues (35) and LINE1-mediated somatic retrotransposition is common in cancer patients (36, 37). Moreover, expression of endogenous LINE1 and other retrotransposons in host cells is commonly up-regulated upon viral infection, including SARS-CoV-2 infection (38–40).

In this study, we show that SARS-CoV-2 sequences can integrate into the host cell genome by a LINE1-mediated retroposition mechanism. We provide evidence that the integrated viral sequences can be transcribed and that, in some patient samples, the majority of viral transcripts appear to be derived from integrated viral sequences.

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