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Previous studies on the role of m\n                  <jats:sup>6<\/jats:sup>\n                  A during HIV-1 replication have produced conflicting results. Since m\n                  <jats:sup>6<\/jats:sup>\n                  A function can vary dramatically by cell type and state, here we aimed to clarify the role of the m\n                  <jats:sup>6<\/jats:sup>\n                  A machinery during HIV-1 replication in primary CD4+ T cells. Using CRISPR-Cas9 we targeted 46 cellular genes implicated in m\n                  <jats:sup>6<\/jats:sup>\n                  A or 5-methylcytosine (m\n                  <jats:sup>5<\/jats:sup>\n                  C) regulation and measured subsequent HIV-1 replication in primary CD4+ T cells. Only knockout of the m\n                  <jats:sup>6<\/jats:sup>\n                  A writer complex auxiliary proteins VIRMA and WTAP, and the m\n                  <jats:sup>6<\/jats:sup>\n                  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\n                  <jats:sup>6<\/jats:sup>\n                  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\n                  <jats:sup>6<\/jats:sup>\n                  A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>IMPORTANCE<\/jats:title>\n                  <jats:p>\n                    m\n                    <jats:sup>6<\/jats:sup>\n                    A is the most common chemical modification on cellular and viral RNA and regulates its stability, localization, and translation. m\n                    <jats:sup>6<\/jats:sup>\n                    A modification and its regulation varies dramatically between cell types and cell states. In this study, we investigated the role of m\n                    <jats:sup>6<\/jats:sup>\n                    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\n                    <jats:sup>6<\/jats:sup>\n                    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\n                    <jats:sup>6<\/jats:sup>\n                    A levels. Our findings suggest regulation by auxiliary members of the m\n                    <jats:sup>6<\/jats:sup>\n                    A writer complex is more influential than the function of the catalytic core itself on HIV-1 infection in primary CD4+ T cells.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.11.07.686373","type":"posted-content","created":{"date-parts":[[2025,11,9]],"date-time":"2025-11-09T01:45:13Z","timestamp":1762652713000},"source":"Crossref","is-referenced-by-count":1,"title":["Impact of N\n                  <sup>6<\/sup>\n                  -methyladenosine (m\n                  <sup>6<\/sup>\n                  A) machinery on HIV-1 replication in primary CD4+ T cells"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8765-1388","authenticated-orcid":false,"given":"Kathryn 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