m6A Methylase and Histone Arginine Methylation as Druggable Targets of SARS-CoV-2

With over two hundred million cases of infected SARS-CoV-2patients reported as of August 2021, COVID-19 continues to be a global dilemma since first identified at the end of 2019. While vaccination efforts have been implemented to curb infection and transmission, the risk of viral spread from fully vaccinated individuals with “breakthrough” cases and the present surge in the highly infectious Delta variant are causes for concern. As such, alternate routes toward stemming SARS-CoV-2 infectivity and transmissibility are being explored that are predicated on epigenetic processes governing the viral life cycle.

Post-transcriptional modifications are known to play a crucial part in the life cycles of certain viruses like human coronavirus. Adenosine methylation in particular, such as m6A, has been reported to affect the viability of specific RNA viruses by modulating viral cap structures, viral reproduction, innate sensing pathways, and the innate immune response [1]. The RNA genome of SARS-CoV-2 contains more than 50 potential m6A sites based on the presence of specific sequence motifs for m6A modification by the RNA methylase complex METTL3/14, including GGACU(T), GGACA, and GGACC. Consequently, greater than 0.64% of all adenosines or 0.18% of all bases, in SARS-CoV-2 RNA could be m6A.

Gain or loss of m6A can result in significant functional changes to RNA viruses, altering host cell fusion/entry, replication, transmission, pathogen intensity, and immune evasion. Results from recent METTL3 inhibitor and depletion studies demonstrating epigenomic suppression of SARS-CoV-2 are indeed encouraging, and suggest m6A methylaseas a viable druggable target for remedial intervention.

Knockdown of host cell METTL3 was shown to decrease SARS-CoV-2 m6A RNA methylation, viral load, percentage of infected cells, infectious virus production, and gene expression of proviral host factors such as ACE2 and TMPRSS2 [2,3]. The reduction of viral genome m6A levels enhanced viral recognition by the innate immune system (RIG-I binding to SARS-CoV-2 RNA), subsequently activating downstream innate immune signaling pathways and inflammatory gene expression [2].

SARS-CoV-2 N protein methylation presents another epigenetic target of interest. N protein interaction with the 5′ UTR of SARS-CoV-2 genomic RNA is required for viral genome packaging. The N protein also serves as a line of defense against the host antiviral response by localizing to and disassembling cytoplasmic stress granules (SGs), thereby hindering induction of innate immune signaling. New evidence shows that inhibition of PRMT1, an enzyme that methylates N protein at residues R95 and R177, or substitution of these arginines hinders N protein—5' UTR interaction, N protein-mediated suppression of SG formation, and SARS-CoV-2 replication [4].

A unique antiviral mechanism has been previously uncovered involving strategic host-mediated alterations in the genomic architecture of invading DNA viruses that blunt their replication and gene expression [5]. By depositing repressive histone marks such as H3K9me3 onto viral genomic material, host cells can induce a heterochromatic remodeling of the foreign DNA. The structural shift to a more tightly packed arrangement imposed upon the viral DNA accordingly hinders accessibility to host cell factors required by the pathogen for its basic subsistence. Whether a similar histone methylation tactic could be exploited during SARS-CoV-2 infection for therapeutic purposes is still unclear and worth further investigating.

EpigenTek's ELISA-based Epigenase assays provide rapid measures of m6A methylase and demethylase activitylevels from cell/tissue nuclear extracts or purified enzymes in a high-throughput format. A unique m6A substrate is stably coated on the assay wells. Bioactive enzymes from input samples will transfer methyl groups to (methylases), or remove them from (demethylases), the bound substrate. Substrate m6A methylation is subsequently detected by a high-affinity antibody specific for this modification.

Quantitative measurements of methylase/demethylase activity and inhibition (for enzyme inhibitor screening) can be quickly obtained within only a few hours. EpigenTek also offers a selection of related tools, including Epigenase Type I PRMT Methyltransferase Activity/Inhibition Assay Kitfor screening PRMT1 inhibitors and SARS-CoV-2 N protein antibodies, tagged SARS-CoV-2 N proteins, and histone methylation assays, to further aid your COVID-19 research needs.

References:

1. Gonzales-van Horn SR, Sarnow P. Making the Mark: The Role of Adenosine Modifications in the Life Cycle of RNA Viruses. Cell Host Microbe. 2017 Jun 14; 21(6):661-669.
2. Li N, et. al.METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection. Cell Rep. 2021 May 11;35(6):109091.
3. Burgess HM, et al. Targeting the m6A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication. Genes Dev.2021 Jul 1;35 (13-14):1005-1019.
4. Cai T, et. al.Arginine methylation of SARS-Cov-2 nucleocapsid protein regulates RNA binding, its ability to suppress stress granule formation, and viral replication.J Biol Chem. 2021 Jul;297(1):100821.
5. Tsai K, Cullen BR. Epigenetic and epitranscriptomic regulation of viral replication. Nat Rev Microbiol.2020 Oct;18(10):559-570.