The goal of this new R01 proposal is to investigate the novel role and mechanisms of N6- methyladenosine (m6A) modification of HIV-1 RNA in viral infection. The internal m6A modification of cellular RNA is a newly emerging mechanism of post-transcriptional control of gene expression, which is coordinately regulated by three groups of host proteins, including adenosine methyltransferases (writers), m6A demethylases (erasers), and m6A-selective- binding proteins (readers). Binding of m6A-modified cellular RNA by the readers, YTH domain family proteins (YTHDF1, 2, and 3), can significantly affect various aspects of RNA functions during translation. However, it is unknown whether HIV-1 RNA contains m6A modification and whether the modification regulates viral replication in CD4+ T-cells. We recently identified multiple regions of m6A modification in HIV-1 genomic RNA (gRNA) bound by the readers (YTHDF1-3 proteins) in HIV-1-infected cells. Our data showed that the expression levels of the readers can significantly modulated HIV-1 postentry infection in target cells. Moreover, knockdown of the m6A writers or an eraser in virus producer cells significantly affected HIV-1 Gag protein synthesis and viral release, suggesting the importance of m6A modification of HIV-1 RNA in viral production. Thus, we hypothesize that m6A modification of HIV-1 RNA regulates viral replication in cells by engaging the m6A readers, writers, and erasers, which can have negative or positive impacts on different stages of the HIV-1 lifecycle. We will test the overall hypothesis in three specific aims mainly using primary CD4+ T-cells.
Aim 1. To examine the role of HIV-1 RNA m6A modification in viral infection and the mechanisms of m6A reader-mediated viral inhibition;
Aim 2. To investigate the role and mechanisms of the m6A writers in regulating HIV-1 infection;
and Aim 3. To investigate the role and mechanisms of the m6A erasers in regulating HIV-1 infection. Overall impact: Accomplishing these proposed studies will reveal the novel role and mechanisms of m6A modification of HIV-1 RNA in regulating viral infection in cells. Studying the m6A modification of HIV-1 RNA and its interactions with host proteins represents a new area of HIV-1 RNA biology, which can facilitate therapeutic development against HIV-1 infection.

Public Health Relevance

HIV-1 infection is the leading killer worldwide among infectious diseases, incurring 2-3 million AIDS deaths annually. A successful cure for HIV/AIDS is depended on complete understanding of HIV-1 interactions with host cells. Studying the mechanisms by which host proteins interact with HIV-1 RNA will provide new insights into regulation of HIV-1 replication in cells, which can facilitate the development of more effective strategies to eradicate HIV-1 infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56AI127667-01
Application #
9315991
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Conley, Tony J
Project Start
2016-08-01
Project End
2017-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
1
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Ohio State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Kenney, Adam D; Dowdle, James A; Bozzacco, Leonia et al. (2017) Human Genetic Determinants of Viral Diseases. Annu Rev Genet 51:241-263
Kohnken, Rebecca; Kodigepalli, Karthik M; Mishra, Anjali et al. (2017) MicroRNA-181 contributes to downregulation of SAMHD1 expression in CD4+ T-cells derived from S├Ęzary syndrome patients. Leuk Res 52:58-66
Wang, Qiankun; Chen, Shuliang; Xiao, Qiaoqiao et al. (2017) Genome modification of CXCR4 by Staphylococcus aureus Cas9 renders cells resistance to HIV-1 infection. Retrovirology 14:51
Li, Chuan; Wang, Hai-Bo; Kuang, Wen-Dong et al. (2017) Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells. J Virol 91:
Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun et al. (2016) N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. Elife 5:
St Gelais, Corine; Kim, Sun Hee; Ding, Lingmei et al. (2016) A Putative Cyclin-binding Motif in Human SAMHD1 Contributes to Protein Phosphorylation, Localization, and Stability. J Biol Chem 291:26332-26342