COVID-19 Genomics: Oxford And John Hopkins Study Shows That SARS-CoV-2 Genome Possess RNA Secondary Structures That Contributes To Viral Persistence

COVID-19 Genomics: A new genomic research by scientist from University of Oxford, and Johns Hopkins Bloomberg School of Public Health shows the presence of of large-scale internal RNA base pairing in the SARS-CoV-2 genome. This property, termed genome-scale ordered RNA structure (GORS) has been previously associated with host persistence in other positive-strand RNA viruses, potentially through its shielding effect on viral RNA recognition in the cell. Genomes of SARS-CoV-2 were remarkably structured, with minimum folding energy differences (MFEDs) of 15%, substantially greater than previously examined viruses such as hepatitis C virus (HCV) (MFED of 7 to 9%).


 
High MFED values were shared with all coronavirus genomes analyzed and created by several hundred consecutive energetically favored stem-loops throughout the genome. In contrast to replication-associated RNA structure, GORS was poorly conserved in the positions and identities of base pairing with other sarbecoviruses—even similarly positioned stem-loops in SARS-CoV-2 and SARS-CoV rarely shared homologous pairings, indicative of more rapid evolutionary change in RNA structure than in the underlying coding sequences.
 
Sites predicted to be base paired in SARS-CoV-2 showed less sequence diversity than unpaired sites, suggesting that disruption of RNA structure by mutation imposes a fitness cost on the virus that is potentially restrictive to its longer evolution. Although functionally uncharacterized, GORS in SARS-CoV-2 and other coronaviruses represents important elements in their cellular interactions that may contribute to their persistence and transmissibility.
 
The study findings were published in the peer reviewed journal: MBIO (an open access journal published by the American Society for Microbiology.) https://mbio.asm.org/content/11/6/e01661-20
 
Importantly the detection and characterization of large-scale RNA secondary structure in the genome of SARS-CoV-2 indicate an extraordinary and unsuspected degree of genome structural organization; this could be effectively visualized through a newly developed contour plotting method that displays positions, structural features, and conservation of RNA secondary structure between related viruses. Such RNA structure imposes a substantial evolutionary cost; paired sites showed greater restriction in diversity and represent a substantial additional constraint in reconstructing its molecular epidemiology. Its biological relevance arises from previously documented associations between possession of structured genomes and persistence, as documented for HCV and several other RNA viruses infecting humans and mammals. Shared properties potentially conferred by large-scale structure in SARS-CoV-2 include increasing evidence for prolonged infections and induced immune dysfunction that prevents development of protective immunity. The findings provide an additional element to cellular interactions that potentially influences the natural history of SARS-CoV-2, its pathogenicity, and its transmission.
 
There are numerous biological effects of large-scale RNA structure in SARS-CoV-2 and other coronaviruses.
 
Despite the description of GORS in HCV and a range of other positive-strand RNA viruses, little is known about the biological effects of large-scale RNA structure in viral genomes and how it may influence interactions with the cell.
 
Double-stranded RNA (dsRNA) represents a potent pathogen-associated molecular pattern for a variety of pattern recognition receptors (PRRs) such as RIG-I, MDA5, and oligoadenylate synthetases (OASs 1 to 3) https://pubmed.ncbi.nlm.nih.gov/18089727/
 
Internal base pairing in virus genomes possessing GORS might therefore appear to predispose recognition by PRRs. However, duplexes formed in SARS-CoV-2 and HCV RNA are typically interrupted and restricted to consecutive pairing lengths shorter than those recognized by PRRs. https://www.pnas.org/content/112/12/3692.short
 
Indeed, possession of GORS may have the opposite effect in compacting RNA into forms that may be resistant to binding by PRRs or nucleases. Biophysically, structured genomes take on a globular, compacted appearance on atomic force microscopy, and sequences are inaccessible to external probe hybridization, indicating a quite different RNA configuration from unstructured viruses and potentially influencing interactions with the cell. https://jvi.asm.org/content/82/23/11824.short
 
Maintenance of RNA structure is costly in evolutionary terms, since most changes at paired sites, and potentially a proportion at unpaired sites, disrupt RNA folding. In a previous bioinformatic experiment, 5% simulated evolutionary drift of HCV, HPgV, and foot-and-mouth disease virus (FMDV) reduced MFED values of each virus genome by >50%. In the real world, longer-term sequence change in these viruses can occur only in a manner that maintains a relatively fixed level of internal base pairing. The observation that SARS-CoV-2 site diversity was substantially influenced by its predicted pairing provides a further indication of the potential phenotypic costs of RNA structure disruption. https://rnajournal.cshlp.org/content/10/9/1337.short
 
A further uncertainty about the purpose and mechanisms of GORS-associated structures is the as yet unexplained correlation between RNA structure formation and virus persistence. Among many possibilities, the study team has previously suggested that decreased virus recognition by the innate immune system may fail to activate interferon and other cytokine secretion from infected cells, leading to downstream defects in macrophage and T cell recruitment and maturation. These defects may ultimately blunt adaptive immune responses sufficiently to enable virus persistence. The poor T helper functions were associated with proliferation defects and deletions of reactive CD4 lymphocyte cell responses in those with persistent infections (40–42). Downstream impairment of CD8 cytotoxic T cell and antibody responses may originate from this failure of immune maturation. https://rupress.org/jem/article/209/1/61/54596/Broadly-directed-virus-specific-CD4-T-cell
 
https://www.sciencedirect.com/science/article/pii/S1074761317304168
 
https://pubmed.ncbi.nlm.nih.gov/11124845/
 
However the finding that not only SARS-CoV-2, but also all four of the seasonal human coronaviruses possess intensely structured genomes does not square with the previously noted association of GORS with persistence. The human seasonal coronaviruses are considered to cause transient and most often unapparent or mildly symptomatic respiratory infection, notwithstanding the dearth of focused studies on durations of virus shedding and potential sites of replication outside the respiratory tract. Interestingly, repeat testing of individuals with diagnosed NL63, OC43, and 229E infections within 2 to 3 months revealed frequent occurrences of infections with the same virus, >20% in the case of NL63. https://pubmed.ncbi.nlm.nih.gov/29741740/
 
In many cases, infections were by the same clade of virus and often showed higher viral loads than observed at the original time point. These findings were interpreted as evidence for reinfection as described in previous studies, and for some individuals, intermediate samples were obtained and shown to be PCR negative. https://pubmed.ncbi.nlm.nih.gov/2170159/
 
https://pubmed.ncbi.nlm.nih.gov/3008551/
 
However, the findings do not rule out persistence over the 3 months of the sampling interval. The observation of NL63 detection in 21% of follow-up samples in a study group where only 1.3% of individuals were initially infected provides some tentative support for the latter possibility. Even if the result of reinfection, the findings demonstrate that seasonal coronaviruses fail to induce any effective form of protective immunity from reinfection even over the short period after primary infection. This resembles findings for HCV, where a potentially comparable immunological defect leads to those who have cleared infection to be readily reinfected with same HCV genotype . https://pubmed.ncbi.nlm.nih.gov/11988247/
 
https://pubmed.ncbi.nlm.nih.gov/22541630/
 
In nonhuman hosts, coronavirus infections are typically persistent where investigated. These include bovine coronavirus (BCoV) which establishes long-term, asymptomatic respiratory and enteric infections in cows. https://link.springer.com/article/10.1186/s12917-019-1887-8
 
https://www.jstage.jst.go.jp/article/jvms/advpub/0/advpub_18-0050/_article/-char/ja/
 
BCoV is closely related to OC43 in humans and potentially its zoonotic source .Although not longitudinally sampled, MERS-CoV was detected at frequencies of >40% in several groups of dromedary camels, similarly indicative of persistence despite its more frequent clearance in infected humans. Other coronaviruses showing long-term persistence include mouse hepatitis virus, feline calicivirus, and infectious bronchitis virus in birds. Pigs are infected with a range of different coronaviruses of variable propensities to establish persistent infections. Many of the coronaviruses characterized in pigs have arisen in major outbreaks potentially from zoonotic sources, including porcine deltacoronavirus in 2014 from sparrow CoV, and porcine epidemic diarrhea virus in 1971 and swine acute diarrhea syndrome-coronavirus in 2016 from bats. A lack of host adaptation immediately after recent zoonotic spread may contribute to the various outcomes of pig coronavirus infections. Coronaviruses in bats are distributed in the Alpha- and Betacoronavirus genera, widespread, highly genetically diverse, and host specific. Establishing whether infections are persistent in bats is problematic in a standard field study setting. However, high detection rates in fecal samples from bats, including 26% and 24% in large samples of Minopterus australis and Minopterus schreibersii in Australia, 29% in rhinolophid bats in Japan, and 30% in various bat species in the Philippines are strongly indicative of persistence. Overall, coronaviruses clearly have a propensity to persist, although their ability to achieve this may depend on their degree of host adaptation.
 
Turning to recently emerged coronaviruses in humans, the course of SARS-CoV infections can be prolonged, up to 126 days in fecal samples, although little information on persistence was collected before the end of the outbreak. https://pubmed.ncbi.nlm.nih.gov/15504274/
 
MERS-CoV infections are persistent in camels but show variable outcomes in humans with respiratory detection and fecal excretion typically ceasing 3 to 4 weeks after infection onset but with individual case reports of much longer persistence in some individuals. https://pubmed.ncbi.nlm.nih.gov/25448335/
 
https://pubmed.ncbi.nlm.nih.gov/15789222/
 
https://pubmed.ncbi.nlm.nih.gov/25516193/
 
Based on what is known for other coronaviruses, SARS-CoV-2 clearly has the potential for persistence and indeed probably is persistent in its immediate bat source, Rhinolophus affinis. https://pubmed.ncbi.nlm.nih.gov/32015507/
 
 Its current presentation as an acute, primarily respiratory infection may represent the typical course of a recently zoonotically transmitted virus with the potential for future adaptive changes to increases its systemic spread and achieve a degree of host persistence apparent in many animal coronaviruses.
 
Even in the relatively short pandemic period of SARS-CoV-2 6 months after the zoonotic event, relatively long periods of respiratory sample detection and fecal excretion of the virus have been documented, in many cases of greater than 1-month duration.
 
https://www.medrxiv.org/content/10.1101/2020.06.08.20125310v1
 
https://onlinelibrary.wiley.com/doi/full/10.1111/codi.15138
 
https://www.medrxiv.org/content/10.1101/2020.05.21.20108605v1
 
https://www.medrxiv.org/content/10.1101/2020.06.02.20120774v3
 
https://www.medrxiv.org/content/10.1101/2020.06.10.20127837v1
 
These occur in both mild and severe cases of COVID-19 in patients, and without comorbidities or evident immune deficits that may separately contribute to persistence. While the world anxiously awaits how SARS-CoV-2 transmissibility and pathogenicity may evolve in future outbreaks, understanding the mechanisms of postzoonotic adaptation of SARS-CoV-2 to humans is of crucial importance. Interactions of SARS-CoV-2 with innate immune pathways potentially modulated by large-scale RNA structure may represent one element in this adaptive process.
 
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