Scientists Discover That Host miRNAs Contribute To Evolution Of SARS-CoV-2 As Witnessed In Mutations Of NSP4 Proteins

COVID-19 News: The positive-sense RNA genome of the SARS-CoV-2 virus, responsible for the COVID-19 pandemic, is approximately 30,000 nucleotides in length. Since its emergence in December 2019 with the WuhanHu-1 strain, over 10,000 recorded nucleotide mutations have been documented, with an estimated mutation rate ranging from 0.0004 to 0.002 mutations per nucleotide per year.


 Pic Credit:CROCOTHERY-iStock

The GISAID database, which houses over 14 million SARS-CoV-2 sequences, allows for detailed tracking of the virus's evolution.
 
A significant factor shaping the virus is the pressure exerted by neutralizing antibodies on the receptor-binding domain of the Spike (S) protein.
 
However, antibodies are not the only element the virus encounters during infection. The binding of host miRNA to other positive-sense RNA viruses provides considerable evolutionary pressure, as these molecules suppress virus translation, replication, and alter aspects of their pathogenesis.
 
Several studies have predicted interactions between the SARS-CoV-2 single-stranded positive-sense RNA virus and multiple human miRNAs.
 
However, most studies do not consider the virus's ability to evolve and selectively alter its miRNA binding regions, particularly those enriched in binding seeds. Only a few works have analyzed sequences from different Variants of Concern (VOCs).
 
Human genomes encode numerous miRNAs, with the majority showing differential expression in human tissues. Only miRNA species expressed in replication-permissive host cells are relevant to SARS-CoV-2 evolution.
 
The virus predominantly replicates in type 2 alveocytes of the lung in humans, but significant clinical data also indicate the presence of the virus in the intestinal epithelium, which expresses ACE2 and TMPRSS2. Intestinal viral persistence is commonly observed, and it is now recognized that long-term SARS-CoV-2 residence in the human gut supports low-intensity inflammation, contributing to post-COVID or long COVID complications.
 
Over the three years of the pandemic, the SARS-CoV-2 virus has undergone substantial mutations.
 
Due to the degeneracy of the genetic code, many mutations have not resulted in changes in viral protein sequences and are classified as silent. However, silent nucleotide changes have become fixed in the virus genome. The fixation of mutations in evolution can occur by chance or under the influence of selective forces acting upon RNA rather than the encoded proteins. Cellular miRNAs binding to the viral genome could exert such pressure.
 
This study tests the hypothesis that the evolutionary pressure exerted by the binding of miRNA species expressed in human lung and intestinal tissue has shaped the genome of the currently dominant SARS-CoV-2 virus variants globally.
 
The study team discovered that host miRNAs contribute to evolution of SARS-CoV-2 as witnessed in mutations of NSP4 proteins.
 
The study team comprised of researchers from: HSE University-Russia, Sun Yat-Sen University-China, George Mason University-USA, Research Centre for Medical Genetics-Russia, Russian Academy of Sciences and Art Photonics GmbH-Ger many.
 
The study team demonstrate that a significant number of miRNA binding sites locate in the NSP4 region of the SARS-CoV-2 genome, and the intestinal human miRNAs exert evolutionary pressure on this region.
 
Notably, in infected cells, NSP4 promotes the formation of double-membrane vesicles, which serve as the scaffolds for replication-transcriptional complexes and protect viral RNA from intracellular destruction. In three years of selection, the loss of many miRNA binding sites, in particular, those within the NSP4, has shaped the SARS-CoV-2 genomes to promote the descendants of the BA.2 variants as the dominant strains and define current momentum of the pandemics.
 
The study findings were published on a preprint server and is currently being peer reviewed.
https://www.biorxiv.org/content/10.1101/2023.03.31.535057v1
 
In this study, the hypothesis that host cell miRNAs may have influenced the evolution of SARS-CoV-2 was tested. The researchers focused on two sets of miRNAs expressed in the main sites of virus reproduction: the lungs and the intestine.
 
These sites were found to have significantly different representations of individual miRNAs. Lung-specific miRNAs included hsa-miR-143-3p, hsa-miR-21-5p, hsa-miR-22-3p, and hsa-miR-30a-5p, with miR-143-3p being highly predominant (41.5%). In contrast, the intestine had a more uniform profile, featuring tissue-specific let-7b-5p, miR-92a-3p, and miR-200c-3p.
 
The study showed that, throughout the pandemic, SARS-CoV-2 genomes lost many miRNAs binding sites specific to lung and intestinal tissue-expressed miRNAs. Monte-Carlo simulations demonstrated that the observed changes were significant only for the set of miRNAs expressed in intestinal tissue (p ≤ 2.50·10−2).
 
Coronaviruses are typically perceived as Acute Respiratory Infections (ARIs), with viral production in the lungs providing a reservoir for virus spread. However, SARS-CoV-2 can also replicate in other organs and tissues, including the intestine, where it can reside for up to three months. This prolonged replication period allows evolutionary forces to act on the virus population within the host, with host factors potentially driving this evolution. Host miRNA binding may represent such a force.
 
Resultant viral populations may re-enter the circulating pool through the fecal-oral route or during secondary viremia in immunocompromised hosts capable of supporting internal reinfection of the lungs and subsequent airborne spread.
 
The Omicron variant initially shared high similarity with variants found in rodents, particularly mice. This led to the hypothesis That was covered in past COVID-19 News reports that an ancestral variant of SARS-CoV-2 entered the rodent population and evolved within these animals before reemerging in humans.
 
https://www.thailandmedical.news/news/breaking-china-s-scientists-say-omicron-most-probably-originated-from-a-mouse-and-not-from-a-human-host
 
An alternative theory suggests long-term virus evolution within an immunocompromised patient, predominantly in the intestines.

This study underscores the importance of the intestinal site in COVID-19 pathogenesis. While pneumonia and respiratory complications account for most morbidity and mortality, extrapulmonary manifestations include diarrhea, nausea, vomiting, anorexia, abdominal pain, and heartburn. These complications occur in 10%-20% of COVID-19 patients, which does not exclude the possibility of asymptomatic gastrointestinal infections with SARS-CoV-2. Latent persistence of the virus in the intestine may contribute to long COVID.
 
A dramatic decrease in miRNA binding sites was particularly pronounced in BA.2-like SARS-CoV-2 variants. This decrease was largely due to mutations in the region encoding the NSP4 protein, which promotes double-membrane vesicle formation for replication-transcriptional complexes and protects viral RNA from intracellular immunity. Along with significant changes in the S-protein, miRNA-driven alterations of NSP4 acquired during prolonged replication within the intestine may have contributed to the global dominance of BA.2 variant descendants.
 
These findings highlight the potential impact of intestinal tissue on the evolution of the SARS-CoV-2 genome and its role in long COVID.
 
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