Study of Human T-Lymphotropic Virus type 2 mRNA kinetics of expression and identification of a novel splicing site

The analysis of HTLV expression during the infection process is essential to understand the influence of viral gene products on proliferation, cell cycle and signalling. Recent studies carried out on HTLV-1-infected cells have provided information on the relative abundance and timing of expression of different viral transcripts. In the case of HTLV-2, very little information has been so far obtained on the profile of viral gene expression in infected cells, and the kinetics of transcript expression have not yet been investigated. The aim of the study was to further investigate HTLV-2 transcripts expression, by developing new quantitative analyses to measure the levels of expression of different HTLV-2 mRNAs. In particular the kinetics of HTLV-2 transcripts at different stages of virus gene expression and their quantitation in infected cell lines and in PBMCs from infected subjects, have been evaluated. Similarly to other retroviruses, HTLV-2 expresses multiple gene products from the same coding region by choosing different strategies, including a complex pattern of alternative splicing. HTLV-2 expression produces three major classes of mRNAs: unspliced mRNA for Gag, Protease and Pol proteins; singly spliced mRNAs for Env and accessory proteins p28 and p22/p20; and doubly spliced mRNAs for the regulatory Tax, Rex and the accessory p10, p11 and 1-2-B proteins. More recently, the new APH-2 protein coded by the negative strand of HTLV-2, and possibly involved in the transcriptional silencing of the virus in infected cells was described. The expression profile and kinetics of the different transcripts were analysed by RT-PCR assays using a series of splice-junction-specific primers and probes. The time courses of each HTLV-2 transcript in three different cellular systems, namely stably infected cells lines, transiently transfected cells and ex-vivo IL-2 stimulated PBMCs from infected subjects were investigated. The results obtained led to the quantitation of all known HTLV-2 mRNAs. Preliminary data showed different levels of env transcript in HTLV-2 subtypes A and B. Evidence for a differential env expression in HTLV-2B stably infected cells, prompted further investigation on the complex pattern of splicing between exons 1 and 2 and allowed the identification of a novel 3’ acceptor site (SS) of splicing. This novel 3’SS is used to express alternative spliced isoforms within the pX terminal region of HTLV-2, including the singly spliced env and the three doubly spliced bicistronic tax/rex, p10/p11 and 1-2-B transcripts. Results demonstrated that the novel 3’SS was utilised in both HTLV-2 subtypes, and that the novel env isoform, named env 1-2b, was preferentially expressed in 2B subtypes, while 2A subtypes used more efficiently the canonical splice site. The kinetics of total mRNA HTLV-2 expression in these three cellular systems presented a general pattern characterised by an initial low transcript level and a subsequent increase to reach a peak of expression after 21-24 hours in culture. The full length gag/pol mRNA was the most abundant one during the time course, and behaved as a late gene that peaked after 21 to 48 hours. The canonical env transcript was expressed at very low rates in stably infected cells, and it was undetectable in PBMCs from infected subjects and in cells transfected with the HTLV-2 proviral clone. By contrast, env 1-2b isoform was efficiently expressed in stably infected cells as well as in ex-vivo infected PBMCs, behaving as a late gene showing a gradual and steady increase in copy number and reaching maximum expression at 21 to 48 hours. The regulatory tax/rex gene was expressed early and with a high/intermediate transcription rate in HTLV-2B subtypes in both stably infected cells and ex-vivo PBMCs. In this study the kinetics of expression of the yet unkwnon 1-2-B gene was investigated and found to be expressed at high levels at early time points, whereas the doubly spliced mRNA of accessory p10/p11 genes were poorly expressed or under detection limit. Among the singly spliced mRNAs coding for other accessory proteins, the p28, p22/p20-II isoform was found to be highly expressed in both HTLV-2 A and B subtypes as compared to its alternative p28, p22/p20-I form. The APH-2 negative transcript was efficiently expressed at high levels in both stably infected and transfected cells and behaved as a late gene. However, in ex-vivo PBMCs its expression level and kinetics pattern appeared to be variable and a clear pattern of expression was not assessed. In conclusion, this study demonstrated that HTLV-2 transcripts of both A and B subtypes are differentially expressed. A temporal pattern of mRNA production, with early, tax/rex and 1-2-B and late, gag/pol, env, p28,p22/p20-II genes was established. The tax/rex early expression indicates that this protein is necessary at the beginning of the viral cycle to transactivate and regulate viral and cellular genes. Most structural genes were expressed late when the transcription of early genes was already decreasing, thus showing that a temporal switch was occurring between early to late genes production. This study also provided new clues on the selective use of alternative 3’SS within the pX region of HTLV-2 for both subtypes A and B. Overall these findings indicate that the control of HTLV-2 viral gene expression is highly regulated both in its kinetics and expression. Moreover, it suggests that the use of multiple acceptor sites might play an important role on the preferential expression of specific proteins in the different phases of the viral cycle.