While the importance of chemical modifications of DNA and proteins is well established, relatively little is known about how the post-transcriptional modification of mRNA transcripts affects their function. The most common modified base seen on cellular mRNAs in mammals is N6-methyladenosine (m6A), and recent data demonstrate that the total loss of m6A formation can have severe deleterious consequences, for example, blocking the differentiation of pluripotent stem cells. However, how m6A regulates mRNA expression remains to be established. Moreover, previous work has revealed that several pathogenic human viruses, including influenza A virus (IAV), encode RNAs that undergo m6A editing, suggesting that such modifications might exert a positive effect on virus replication. Indeed, recent data from this laboratory, and others, demonstrate that m6A editing can significantly enhance HIV-1 gene expression and replication in cultured T cells. This grant proposal seeks to fully define the sites of m6A modification on IAV transcripts and to test the hypothesis that m6A editing enhances IAV gene expression and, hence, replication. Using the PAR-CLIP technique, we have identified several specific binding sites for the human YTHDF m6A reader proteins on the IAV genome on both the viral mRNA/cRNA and vRNA transcripts. Consistent with the hypothesis that m6A editing can positively regulate IAV gene expression, we have observed that overexpression of the human YTHDF2 reader protein, but not of the related YTHDF1 protein, in human A549 cells greatly enhances IAV replication. This grant first aims to define the precise locations of m6A modifications on IAV transcripts at single nucleotide resolution and to then quantify the degree of editing at each site. We will then use targeted mutagenesis to determine precisely how specific m6A modifications on different segments of the IAV genome affect IAV gene expression and replication. In parallel, we will examine how the overexpression of key cellular proteins involved in ?writing?, ?reading? and ?erasing? m6A marks, or their elimination by genome editing using CRISPR/Cas, affects IAV gene expression and replication in A549 cells. Together, these data will reveal how m6A editing affects IAV replication and may lead to new approaches to the enhancement of IAV replication in culture, for use for example in IAV vaccine production, or the inhibition of IAV replication in vivo, for example by transient inhibition of m6A editing.

Public Health Relevance

Influenza A virus (IAV), an important human pathogen, encodes RNAs that are modified by addition of methyl groups to the N6 position of specific adenosine residues. The purpose of this proposal is to identify which adenosine residues are modified and to test whether these modifications affect IAV replication, as suggested by our preliminary data.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI130574-01
Application #
9296268
Study Section
Virology - A Study Section (VIRA)
Program Officer
Hauguel, Teresa M
Project Start
2017-01-06
Project End
2018-12-31
Budget Start
2017-01-06
Budget End
2017-12-31
Support Year
1
Fiscal Year
2017
Total Cost
$238,500
Indirect Cost
$88,500
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Kennedy, Edward M; Bogerd, Hal P; Kornepati, Anand V R et al. (2017) Posttranscriptional m6A Editing of HIV-1 mRNAs Enhances Viral Gene Expression. Cell Host Microbe 22:830
Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E et al. (2017) Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication. Cell Host Microbe 22:377-386.e5
Kennedy, Edward M; Courtney, David G; Tsai, Kevin et al. (2017) Viral Epitranscriptomics. J Virol 91: