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   Home  »  Epigenetic Resources  »  m6A May Help Inhibit Viral Replication 
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m6A May Help Inhibit Viral Replication

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Cellular metabolism is a set of complex chemical reactions responsible for maintaining life. When a virus infects a cell, it hijacks the cellular metabolism and uses it to grow and spread. Typically, when a cell recognizes an invading virus, it either shuts it’s metabolism down so the virus cannot replicate, or it will repurpose its existing machinery to ensure viral survival.

N6-methyladenosine (m6A) is the most common RNA modification, and it is present in all eukaryotes. Modified adenosines like m6A can affect the health and success of a virus by regulating viral gene expression, sensing pathways, and the innate immune response.

In a study out of Peking Union Medical College in China, a team of researchers set out to determine the specific role that m6A modifications play in viral replication in mice, and in human monocytic cells called THP-1 cells.

The team, led by Dr. Yang Liu, conducted this study using healthy mice and mice deficient in a commonly known RNA demethylase called ALKBH5. The mice were infected with either vesicular stomatitis virus (VSV) or Herpes simplex virus 1(HSV-1), then monitored for m6A levels every 4 hours for 24 hours.

The team extracted nuclei from the mice using the EpiQuik Nuclear Extraction Kit. They then used to Epigenase m6A Demethylase Activity/Inhibition Assay Kit to measure the activity of ALKBH5 in the extracted nuclei. Upon infection, they saw an initial increase in m6A in the first 12 hours. As the incubation period progressed, m6A levels began to decrease in tandem with the presence of the virus.
m6A abundance in VSV infected cells
(A) Western blot of VSV-G protein (top) and Dot blot of m6A levels (250 ng total RNA, middle) in mouse peritoneal macrophages infected with VSV for indicated times, and the m6A level intensity (bottom)


The ALKBH5-deficient mice demonstrated significantly lower VSV reproduction, suggesting that the demethylation of m6A can promote viral growth. The researchers confirmed their findings by using CRISPR-Cas9 to knockout ALKBH5 both in the mouse cells and in THP-1 cells. Both of these knockouts resulted in a decrease in virus production in the cells.

Next, the team wanted to determine which metabolic gene functions were altered in ALKBH5-deficient cells after infection. They specifically focused on two major metabolic components: a gene called Oxoglutarate Dehydrogenase (OGDH), an important catalyst in regulating the Krebs cycle, and a chemical compound called Itaconate that is metabolized during immunity.

In the ALKBH5-deficient cells, they discovered that both OGDH and Itaconate had been significantly down-regulated, resulting in halted viral reproduction. They performed a knockout of OGDH in infected mouse cells and THP-a cells and found that Itaconate was reduced, once again resulting in inhibited viral reproduction.

OGDH in ALKBH5 KO macrophages
(A) qPCR (top) and Western blot (bottom) of OGDH in ALKBH5 KO macrophages infected with VSV..


To determine if an absence of ALKBH5 increased m6A methylation levels, the team ran a transcriptome-wide m6A profiling of infected normal and infected ALKBH5 deficient cells. They were able to identify overlapping peaks between OGDH and ALKBH5, and also found that they both had decreased in the infected cells, suggesting that ALKBH5 can demethylate OGDH and help regulate virus replication

Not much was previously known about these pathways and how they respond to viral infection. But the findings in this study suggest that when a cell encounters a virus, it will inhibit ALKBH5 activity, leaving m6A to limit viral reproduction. OGDH and Itaconate were found to promote viral replication in host cells. However, m6A effectively down-regulate these two factors and halted viral replication, identifying OGDH and Itaconate as potential targets for the treatment of viral infections.

Reference:
Liu, Y et. al. (2019). N6-methyladenosine RNA modification–mediated cellular metabolism rewiring inhibits viral replication. Science. 1171-1176.

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