HIV-1 Infection Alters Post-Transcriptional Processing of HTLV-1 RNA in MT-2 Cells

Human T-lymphotropic virus type 1 (HTLV-1) gene expression is extensively regulated at the post-transcriptional level, but how human immunodeficiency virus type 1 (HIV-1) infection affects the processing of HTLV-1 RNA remains unclear. Here, we combined reverse transcription quantitative PCR (RT-qPCR) with Oxford Nanopore direct RNA sequencing (ONT DRS) to characterize HTLV-1 RNA levels, alternative splicing, poly(A)-tail length, and RNA modifications in HIV-1-infected MT-2 cells. HIV-1 infection did not significantly alter total HTLV-1 RNA levels but was associated with changes in HTLV-1 RNA processing. D1-A8 splicing became predominant, and the proportion of p21rex transcripts increased from 29.45% to 82.07% of total HTLV-1 transcripts. Poly(A)-tail changes were transcript-specific, with longer tails in env and shorter tails in p21rex, HBZ, and unannotated transcripts. HIV-1 infection also altered N6-methyladenosine (m6A), 5-methylcytidine (m5C), and pseudouridine (Ψ) modifications, with more extensive changes in m5C and Ψ than in m6A. Demethylation mediated by alpha-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) further supported the assignment of nucleotides 7011, 7357, and 7691 as m6A modification sites. Together, these findings demonstrate that HIV-1 infection alters the post-transcriptional regulation of HTLV-1 RNA despite stable total HTLV-1 RNA levels.