Impact of N 6 -methyladenosine (m 6 A) machinery on HIV-1 replication in primary CD4+ T cells
Year: 2025
Type: preprint
Abstract: ABSTRACT N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of cellular and viral RNA and is critical to the regulation of its localization, stability, and translation. Previous studies on the role of m 6 A during HIV-1 replication have produced conflicting results. Since m 6 A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m 6 A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m 6 A or 5-methylcytosine (m 5 C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m 6 A writer complex auxiliary proteins VIRMA and WTAP, and the m 6 A reader YTHDF2 were validated as significantly decreasing HIV-1 replication. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in only marginal changes in HIV-1 infection, despite significant decreases in total cellular m 6 A levels. Chemical inhibition of METTL3 led to a dose-dependent decrease in HIV-1 infection, coupled with an increase in protein levels of METTL3 and other writer complex members. Expression of writer proteins was also co-dependent, revealing complex regulatory feedback mechanisms. Overall, these results clarify the role of epitranscriptomic machinery during HIV-1 replication in primary CD4+ T cells and suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells. IMPORTANCE m 6 A is the most common chemical modification on cellular and viral RNA and regulates its stability, localization, and translation. m 6 A modification and its regulation varies dramatically between cell types and cell states. In this study, we investigated the role of m 6 A factors during HIV-1 infection of physiologically relevant primary CD4+ T cells. Using CRISPR-Cas9 to knockout 46 cellular genes implicated in RNA modification, we found only the m 6 A writer complex auxiliary members WTAP and VIRMA, and the reader YTHDF2, significantly affected HIV-1 replication in these cells. In contrast, knockout of METTL3 or METTL14, which form the catalytic core of the writer complex, resulted in marginal changes in HIV-1 infection, despite larger reductions in total cellular m 6 A levels. Our findings suggest regulation by auxiliary members of the m 6 A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.
Authors: Kathryn A. Jackson-Jones, Lacy M. Simons, Siyu Huang, Thea L. Joseph, Aubrey M. Sawyer, Li Wu, Judd F. Hultquist
Institutions: Northwestern University, University of Iowa
Language: English
Topic: RNA modifications and cancer
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Awards: G2746151604, Regulation of SAMHD1 antiviral activity, G4159924833, G4406492294, Functionally Defining HIV-Host Interactions During the Early HIV-1 Lifecycle, G534236435, G619466266, G867868940, G9000026354, G9000168083, G9000189621, G9000213050, Deciphering the Role of CPSF6 in HIV Infection, Targeting HIV-1 RNA modifications in latently infected CD4+ T cells for therapeutic development, HARC: HIV accessory and regulatory complexes, Controlling HIV latency by manipulating CycT1 turnover
OpenAlex ID: W4416124828