Background
N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. The core m6A machinery consists of writers that deposit the modification, erasers that remove it and readers that respond to the presence of m6A on RNA. Previous studies on the role of m6A during Human Immunodeficiency Virus (HIV) infection have produced conflicting results, particularly in regard to which readers influence HIV replication. Due to the significant functional variation of m6A modifications between cell types, we aimed to characterize the role of m6A during HIV infection using primary CD4+ T cells.
Methods
The expression of the core m6A machinery in primary CD4+ T cells was assessed using qPCR assays. Systematic knockout of 46 genes implicated in m6a modification was carried out using a CRISPR-Cas9 system in primary CD4+ T cells. Edited cells were subjected to a spreading infection using a HIV-1 NL4-3 GFP reporter virus; and subsequently screened by flow cytometry to quantify infection levels. We then carried out m6A-methylated RNA immunoprecipitation sequencing (meRIP-seq) to map m6A sites transcriptome-wide in primary CD4+ T cells upon knockout of core writers.
Results
qPCR interrogation of core m6A machinery in primary CD4+ T cells found that all genes are expressed, and their expression is unaffected by interferon stimulation. Of the 46 genes investigated in the CRISPR-Cas9 knock-out system, only the writer complex, the core erasers, and the reader YTHDF2 significantly altered HIV infection. Analysis of the meRIP-seq data for knockouts of m6a writers resulted in a reduction in m6A sites across the transcriptome and lower amounts of m6A deposited on the HIV genome. The average position of m6a peaks over mRNA’s across the human genome was bias towards the 3’ end of the coding sequence. m6A peaks across the HIV genome were clustered at the 3’ end of env and nef across all conditions; and unique peaks were observed in gag and pol in the METTL3 and WTAP knock-out populations.
Conclusions
Taken together, these studies provide clarity regarding the role of m6A modification machinery in HIV-1 replication in primary CD4+ T cells. Understanding how these modifications are involved in HIV infection, latency, and pathogenesis may lead to the development of next-generation therapeutics targeting viral RNA.
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