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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - IAV-m6A (Elucidating the role of m6A RNA methylation on the replication and pathogenesis of influenza A virus.)

Teaser

Summary

Epitranscriptomics is the study of how small marks, or modifications, on RNA molecules affect how they function. The RNA bases are modified by protein \'writers\'. These writer proteins are in both eukaryote and prokaryote cells, which tells us that this process of writing modifications onto RNA has been conserved across billions of years of evolution, hinting at their importance. I am interested in determining exactly what these modifications are doing to the RNA in our cells. One way to study this is by looking at how viruses have evolved to use these modifications. In the 1970s and 1980s research was carried out into 1 specific modification, m6A, on influenza A virus. This m6A modification was found to be quite abundant on the influenza RNA, compared to normal human cellular RNA. However, due to limitations in the technologies at the time no further work was performed to investigate what exactly these m6A modifications do. This project focuses on influenza A virus and aims to locate each m6A on each viral RNA. Once I know the location of each m6A modification I will be able to individually remove these sites from viral RNA and investigate exactly how the characteristics of viral RNA are altered due to this loss. By understanding what these modifications do on viral RNAs I can shed some light on their role on cellular RNAs also. In addition, if I find that these modifications are essential for viruses like influenza A, it may be possible to design antiviral drugs to stop these modifications being added to influenza RNA and thus block infections.

The overall objectives of this work are to; locate where each of these m6A modifications are on influenza A RNAs, define exactly how these modifications affect their target RNA and the cell as a whole, work out whether influenza somehow regulates the amount of modifications happening in a cell after infection, and to check whether these m6A modifications located on 1 strain of influenza A are also found in the same places on different seasonal strains of influenza.

Work performed

Since the beginning of the project I have successfully identified all the regions of m6A modification on the RNA from 1 strain of influenza A virus, PR8. I identified these sites using 2 different methods. One uses an antibody that only recognises the m6A modification on RNA. The antibody binds to the RNA, is crosslinked and then pulled down. Another method makes use of the fact that there are a few cellular proteins that specifically bind to RNA regions that contain an m6A. These proteins have been termed \'readers\' and this is believed to be the main way that m6A changes the characteristics of modified RNA. I investigated exactly where on the influenza RNA these reader proteins bound and found that a lot of these regions aligned with regions that the antibody identified as containing m6A. This allowed me to precisely m6A modifications are on influenza RNA with a high degree of confidence.

Having confirmed that influenza RNAs are highly modified with m6A I next wanted to look at how well influenza grows in cells lacking the key m6A writer protein, or in cells that over express 2 m6A reader proteins. I found that influenza grows much slower in cells lacking the key m6A writer, known as METTL3, and that this is due to having much lower RNA levels. I also found that these cells then produced a lot less virus after they had been infected. I saw only 10% the amount of virus coming from infected METTL3 knockout cells when compared to from normal cells. This indicates that m6A deposition positively regulates influenza A. I then investigated the growth rate of influenza virus in cells that were over expressing 2 important readers of m6A, known as YTHDF1 and YTHDF2. When I made over expression cell lines I found that there was an increase in viral RNA and protein levels, and there was a greater number of influenza virions being released from these infected cells. Having more YTHDF1 doubled the amount of influenza virus being released from infected cells, but by producing more YTHDF2 I found that virus release was 10 times greater than in normal control cells. This perfectly correlates with the data obtained from the METTL3 knockouts. When m6A is absent influenza grows 10 times slower in a cells, and when the levels of the m6A reader protein YTHDF2 are increased that in turn increases influenza growth 10 times.

I then took this a step further, and by making a few silent mutations in the sequence of one of the influenza genes, hemagglutinin (HA), I could stop m6A modifications being added. I took this mutant virus and compared its growth kinetics to that of a normal unmutated influenza virus. I found that this virus, which had only been mutated to lose m6A sites from 1 of its 8 RNAs, had a significantly reduced viral growth rate. In addition, I was able to determine that this was caused by a significant reduction in the expression of HA protein, and specifically due to a reduction in HA RNA levels. The protein expression and RNA levels of each of the other 7 viral genes remained unaffected in the mutant virus, indicating that the effects I saw were indeed due solely to the loss of m6A modifications.

Final results

Due to my experience locating m6A modifications on influenza RNA, I have now been able to develop novel state-of-the-art methods for mapping additional modifications, including m5C, pseudouridine and ac4C. This will be very helpful in trying to determine the presence of additional modifications on influenza RNA. I expect within the next 12 months I will have mapped a number of these modifications on influenza A, which will help increase our knowledge of the role these RNA modification play in RNA biology.

Within this project I published the first description of a virus where viral RNA was silently mutated to be m6A deficient. I then explored the effect this had on the viral life cycle. I found that the m6A deficient RNA, compared to the wild type RNA, did not induce an altered immune response, did not affect packaging and did not affect nuclear:cytoplasmic localisation. I did find that RNA levels were altered and that this correlated with a reduction in protein expression. Therefore I can determine that m6A on influenza RNA does not affect translation, but rather characteristics of RNA. I have not yet determined if the loss of m6A affects RNA stability, half-life or secondary structure.

This work has so far proven that viruses have evolved to positively incorporate RNA modifications, in this case m6A, into their viral RNA. This makes sense, because if there is a benefit to having m6A deposited on RNA, a fast-evolving RNA virus such as influenza would quickly mutate and evolve to maximise this benefit. That would explain why we find more m6A/kilobase on multiple viral RNA when compared to cellular mRNA. As I and others have now determined that m6A plays a significantly positive role in the life cycle of multiple RNA viruses, small molecule drugs can now be developed to transiently reduce the capabilities of cells to deposit m6A, and thereby slow down viral growth. I have already shown that this is possible with a methyl-donor inhibitor 3-deazaadenosine, which blocks the deposition of all m6A. I demonstrated that supplementation of cells with a non-toxic dose of DAA did not impact on cell viability but reduced influenza viral growth and virus production by 10 fold.