Heterogeneity of HTLV-1 proviral integration sites and internal structures in the ATL cell line MT-1

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). The ATL-derived cell line MT-1 is used to study HTLV-1 biology and leukemogenesis. However, the proviral integration sites and internal structures of HTLV-1 are not comprehensively characterized in MT-1 cells. We analyzed two independently maintained MT-1 cell lines (MT-1 J and MT-1 M) using long PCR, quantitative PCR-based proviral load analysis, inverse long PCR, inverse PCR, integration site-specific PCR, direct sequencing, and Rapid Amplification of Integration Site without Interference by Genomic DNA contamination. Long PCR and proviral load analyses demonstrated that MT-1 cells harbor multiple full-length and defective HTLV-1 proviruses. MT-1 J cells exhibited reduced proviral load in the pX region, suggesting the presence of pX-lacking proviruses. Inverse long PCR and inverse PCR revealed at least five proviral integration sites in MT-1 J cells; MT-1 M cells contained three. Site-specific PCR confirmed the differential preservation of integration sites. Sequence analysis revealed two full-length proviruses and three type I defective proviruses with distinct internal deletions, including an unreported provirus with a large deletion encompassing pX. Rapid Amplification of Integration Site without Interference by Genomic DNA contamination identified major proviral clones shared between MT-1 J and MT-1 M cells, while revealing differences in clone frequencies and minor integration sites. The MT-1 cell line is a polyclonal population containing multiple full-length and defective HTLV-1 proviruses. Its proviral composition can change during long-term in vitro passaging. This heterogeneity should be considered when interpreting results obtained using MT-1 cells in HTLV-1 and ATL research.