YTHDF1 and YTHDC1 m ⁶ A reader proteins regulate HTLV-1 <i>tax</i> and <i>hbz</i> activity

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus responsible for adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), a progressive neurodegenerative disease. Regulation of viral gene expression plays a key role in viral persistence and pathogenesis. However, the molecular mechanisms underlying this fine-tuned regulation remain poorly understood. Little is known regarding RNA chemical modifications of HTLV-1 RNA and how these affect viral biology and disease development. Post-transcriptional chemical modification of RNA is common in eukaryotes, with N⁶-methyladenosine (m⁶A) being the most prevalent. In this study, we investigated the role of m⁶A RNA modifications on HTLV-1 gene expression. Using MeRIP-Seq, we mapped the sites of m⁶A modification to the 3' end of the viral genome. We found HTLV-1 RNA, as well as viral oncogene transcripts <i>tax</i> and <i>hbz</i>, contained m⁶A modifications. m⁶A-depletion in HTLV-1-transformed cells decreased sense-derived viral genes (<i>Tax, Gag,</i> and <i>Env</i>) and increased antisense-derived <i>Hbz</i> expression. <i>Tax</i> and <i>hbz</i> transcripts were bound by reader proteins YTHDF1 and YTHDC1 in a panel of HTLV-1 T-cell lines. Using expression vectors and shRNA-mediated knockdown, we found that YTHDF1 had opposing effects on viral gene expression, decreasing sense-derived viral genes and increasing antisense-derived <i>Hbz</i>. Upon further study, the YTHDF1 effects on <i>tax</i> abundance were dependent on <i>tax</i> m⁶A deposition. The nuclear m⁶A reader protein YTHDC1 affected the abundance of both sense- and antisense-derived viral transcripts and specifically enhanced the nuclear export of <i>tax</i> transcript. Collectively, our results demonstrate global m⁶A levels and m⁶A reader proteins YTHDF1 and YTHDC1 regulate HTLV-1 gene expression.IMPORTANCEHuman T-cell leukemia virus type 1 (HTLV-1) persistence and pathogenesis are controlled through tight regulation of viral gene expression. The fate of RNA can be controlled by epigenetic modifications that impact gene expression without altering the DNA sequence. Our study details the impact of N6-methyladenosine (m⁶A) RNA chemical modifications on HTLV-1 gene expression. We found that reductions in global m⁶A levels affected viral gene expression, decreasing <i>Tax</i> and other sense-derived viral genes, whereas increasing the antisense-derived <i>Hbz</i>. Our results suggest the oncogenic viral transcripts, <i>tax</i> and <i>hbz</i>, are m⁶A-modified in cells. We found that these viral RNA modifications are interpreted by reader proteins YTHDF1 and YTHDC1, which dictate the fate of the viral RNA. Understanding HTLV-1 RNA chemical modifications offers potential insights into novel therapeutic strategies for HTLV-1-associated diseases.