BREAKING! Scientists Discover Non-Canonical RNA Secondary Structure: G-Quadruplex (RG4) In Monkeypox Strains That Are Currently Spreading Globally!

Monkeypox Research: Scientists from the Center for BioAnalytical Chemistry, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing-China have made a shocking discovery that the current monkeypox strains that are circulating globally contain a non-canonical RNA secondary structure ie a G-Quadruplex (RG4)


 
Non-canonical RNA segments are formed as a result of secondary structures. These include G-quadruplexes, triplex forming oligos, hairpins and cruciforms, and i-Motif structures. RNA secondary structures are involved in the regulation of mRNA function and its fate besides playing important roles in protein structure and protein folding that also affects pathogenesis.
 
From 1 Jan 2022 to 12 July 2022, more than 10,064 cases of monkeypox have been reported in 67 countries with almost 78 percent of the cases just in Europe. This unusual outbreak of monkeypox has raised new concerns in academia and the public.
 
In order to keep abreast of the trend of the monkeypox epidemic, it is extremely urgent and important to surveille the accumulated genomic mutations and the change of the transmission ability of the pathogen, monkeypox virus (MPXV).
 
The Monkeypox Research team from China discovered a non-canonical RNA secondary structure, G-quadruplex (RG4), that surprisingly evolved stepwise with various variants.
 
This RG4 motif is located in the coding sequence region of MPXV C9L gene that is functional in inhibiting host innate immune response. The evolution decreases the stability of this RG4 and promotes C9L protein level in living cells. Importantly, all the reported MPXV genomes in 2022 contain the most unstable RG4 variant, which may be the reason of the increasing spread of MPXV.
 
The study team recommends that health authorities and researchers pay attention to the genomic evolution of MPXV.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2022.06.18.496696v1
 
The study described the evolutionary profile of the monkeypox virus.
 
It is the first study to report a non-canonical ribonucleic acid (RNA) secondary structure called the G-quadruplex (RG4) present in the circulating MPXV variants that are causing the current monkeypox surge globally.
 
The study team employed a quadruplex forming G-rich sequence (QGRS)-mapper to assess the putative G4-forming sequences (PQSs) present in the MPXV genome.
 
As the current monkeypox strains in circulation have accumulated various mutations across different variants, the study team obtained 177 complete viral genomic sequences corresponding to the MPXV genome.
 
These monkeypox viral genomic sequences were used to predict the varied PQSs present in the viral strains and analyze the magnitudes of sequence conservation among the PQSs.
 
The detailed evolutionary relationship between the different C9L PQSs found in the various MPXV strains was identified by analyzing the phylogeneti c tree as per the 177 whole-genome deoxyribonucleic acid (DNA) sequences of MPXV.
 
The generation and stability of the RG4 structures present in the nine C9L PQSs were assessed by conducting a fluorescent turn-on assay using a thioflavin T (ThT), which is a G4-specific fluorescent probe.   
                       
The study findings showed that out of all the MPXV genome sequences, only one PQS was located in the region corresponding to the coding sequences (CDS) in the C9L gene. The C9L gene in the MPXV encodes a Kelch-like protein that is essential in inhibiting innate immune responses related to the host.
 
The study team noted five to eight G-tracts in nine variants corresponding to C9L PQSs. Some of the C9L PQS variants displayed G-tracts with three G-bases, while others had two G-bases.
 
Also, the C9L PQSs related to the different viral strains displayed diverse G-scores as well as overlaps, which suggested varied stability and potential for the formation of G4. Five G-tracts present in the C9L PQSs were found to be highly conserved within the MPXV genome, suggesting a potential function in the viral life cycle of MPXV.
 
The study team also observed that MPXV has two primary genetic clades, the Western African clade, and the Central African clade, along with other subclasses. All the genomic sequences detected in the MPXV cases were significantly related to each other and belonged to the West African B.1 clade.
 
The MPXV genomes found in the A.1, A1.1, and B.1 clades contained C9L-RG4-5, which is the PQS having the least number of G-tracts while the rest of the clades had PQSs having more G-tracts. This suggests that the C9L RG4 has an evolutionary function.
 
In the study, the fluorescent assay with thioflavin T showed that the annealing of the C9L PQS oligos led to an increase in the fluorescent signal of ThT. On the other hand, the G-A mutation of the G-tracts of the C9L PQS did not increase the fluorescent intensity.
 
These findings suggested that the nine C9L PQSs could effectively form G4 structures within a general cytosolic condition. Furthermore, the C9L PQS oligos displayed varied fluorescent intensity at the peak wavelength at the same concentration as ThT, suggesting that the mutations accumulated in the C9L G4 motifs regulated the stability of the RG4 structures.
 
The study team furthermore observed that the stability of the C9L RG4 was higher in the Central African clade than in the West African clade. The strains related to the West African clade comprise the most unstable motif called C9L-RG4-5, while the strains belonging to the Central African clade primarily contain the comparatively more stable motif called C9L-RG4-7-2G3.
 
The researcher further noted that the RG4 motifs belonging to the MPXV strains that have similar evolutionary distances also have similar stabilities.
 
Furthermore, the highly stable C9L RG4 motifs, including C9L-RG4-8-2G3, C9L-RG4-8-3G3, and C9L-RG4-8-5G3 were detected in only a few strains, which indicated a low contagion.
 
Thse study findings indicated that MPXV evolution was likely to reduce C9L-RG4 stability and thus boosted the spread of MPXV.
 
Importantly, the RG4 structure present in the CDS region was also found to effectively inhibit the translation of proteins according to the stability of the RG4 structure.
 
As the C9L gene is associated with the inhibition of host immune response, the study team inferred that the genetic evolution of C9L-RG4 could control the activity of MPXV by enhancing the C9L protein level. This suggested that improved MPXV transmission is correlated to C9L-RG4 evolution.
 
In conclusion, the study team said that the findings showed that MPXV evolution leads to the generation of increasingly unstable C9L RG4 motifs while the current MPXV strains have shown the presence of the highly unstable C9L-RG4-5 motif.  
 
Thailand Medical News would like to add that the study findings do not bode well especially in the current COVID-19 pandemic with billions of people exposed to the SARS-CoV-2 virus are having a state of immune dysfunction or even COVID-19 induced immunodeficiency. https://www.thailandmedical.news/news/breaking-italian-study-validates-previous-claims-that-sars-cov-2-infections-causes-immunodeficiency-conditions-worse-than-hiv-infections
 
Having another viral pathogen that is also able to affect the human host innate immune system could further complicate and compound various health issues.
 
For the latest Monkeypox Research, keep on logging to Thailand Medical News.