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Elucidating the role of m6A RNA methylation on the replication and pathogenesis of influenza A virus.

Periodic Reporting for period 2 - IAV-m6A (Elucidating the role of m6A RNA methylation on the replication and pathogenesis of influenza A virus.)

Período documentado: 2019-06-01 hasta 2020-05-31

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. I can also use these unmodified viral RNAs to identify whether any cellular RNA-binding proteins specifically only bind regions of RNA containing modifications. 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 result in the specific recruitment of some cellular RNA binding proteins to the site of modification.
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. 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 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.

I then made a few silent mutations in the sequence of one of the influenza genes, hemagglutinin (HA), to 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 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.

During my incoming phase I proceeded to try and identify RNA binding proteins that are specific to m6A modified IAV RNAs. I optimised a protocol known as eRIC, originally published by the Hentze group in Heidelberg, to specifically capture an individual species of IAV RNA, namely the NP mRNA, from infected A549 cells. This protocol was successfully optimised, and towards the end of my fellowship I managed to generate a dataset of all cellular RNA binding proteins that specifically associate with NP mRNA during the early phases of the IAV replication cycle in A549 cells. I plan on exploring this technique further and its use for identifying RNA modification reader proteins in my own independent lab, which I am establishing at Queen’s University Belfast.

The overall results of this project are that m6A modifications are highly prevalent on both IAV plus sense mRNAs and anti-sense vRNAs. These modifications positively regulate the viral replication cycle, and upon silent mutagenesis to prevent the addition of these modifications to just a single IAV segment RNA, in this case HA, the progeny virus was found to be significantly attenuated in both cell culture and in vivo mouse infection models. I presented the work performed as part of this fellowship at 4 international scientific conference, as a guest lecturer at the North Carolina School of Science & Math, at the RNA 2017 Satellite workshop and have received invitations to speak at 2 European research institutes on this work at the end of 2020. This research contributed to 5 research publications in high impact scientific journals, and also provided proof of concept for my successful application for an ERC Starting Grant.
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.
Shows the affect of m6A machinery on influenza A production