Complex pattern of HTLV-2 splicing and expression in PBMCs from infected patients

Abstract

HTLV express multiple gene products from the same coding region by employing different strategies, including alternative splicing. Our investigations were focused on the analysis of the levels of expression of different HTLV-2 transcripts, to test their temporal expression at different stages of viral cycle and their quantitation by real time RT-PCR, in infected cell lines and in primary cultures of PBMCs from infected subjects. An early transcription of tax/rex regulatory mRNA was observed, followed by a gradual and steady increase of gag/pol and env structural transcripts and of other accessory mRNAs. We identified a novel 3\u2019 splice acceptor site, used by both HTLV-2 A and B subtypes to generate alternative doubly spliced mRNAs within the pX region and also a novel env isoform preferentially expressed in 2B subtypes and behaving as a late gene. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed as compared to its alternative p28, p22/p20-I form. The APH-2 transcript from the negative strand behaved as a late gene in stably infected cells, while in ex-vivo PBMCs its kinetics appeared to be variable so that a clear pattern of expression was not yet assessed. In conclusion, the temporal transcription of different HTLV-2 transcripts follows a distinct expression pattern: tax/rex is the first mRNA to be expressed, thus indicating that it is necessary at the beginning of the infection cycle to transactivate and regulate viral and cellular transcripts, while gag/pol and env structural genes are expressed later in the viral cycle