Quantitative analysis of Human T-Lymphotropic Virus Type 1 (HTLV-1) gene expression using nucleo-cytoplasmic fractionation and splice junction-specific real-time RT-PCR (qRT-PCR)

Like other complex retroviruses such as HIV-1, HTLV-1 encodes several regulatory and auxiliary non-structural proteins from overlapping open reading frames through the generation of alternatively spliced mRNAs. HTLV-1 expression is orchestrated by the Tax and Rex regulatory proteins; Tax drives the transcription of the viral genome, while Rex acts at the posttranscriptional level by enhancing the nuclear export and expression of unspliced and incompletely spliced mRNAs. The present chapter is focused on the techniques employed to quantitate HTLV-1 mRNAs in the nuclear and cytoplasmic compartments. To ensure a quantitative transcript-specific detection of the levels of individual HTLV-1 mRNAs in a complex mixture of closely related species, splice junction-specific primers and TaqMan probes were used. As HTLV-1 gene regulation is based on the controlled nucleo-cytoplasmic export of the different viral mRNAs, we quantitated the individual viral transcripts in the nuclear and cytoplasmic fractions.