Analisi dell'espressione del retrovirus "Human T-cell Leukemia Virus" di tipo 2 (HTLV-2) in linee cellulari infettate e in linfociti di pazienti

The research activity of this doctoral thesis was focused on two different projects. The first one concerned the study of the transactivating proteins Tax of the human retrovirus HTLV (Human T Cell Leukemia Virus), and more in detail by comparing Tax-1 and Tax-2B proteins, of HTLV-1 and HTLV-2B respectively, and analysing post-translational modifications of Tax-2B. The second project was focused on the analysis of the expression levels of the different transcripts of HTLV-2. The first research project was centred on the study of Tax proteins, given their responsibility for. To highlight possible functional differences between Tax-1 and Tax-2B, which are considered responsible for the different pathogenicity of the two viruses (Lewis et al., 2002; Feuer e Green, 2005), a careful comparison was undertaken. To this end, a new detection system for the two proteins, based on the insertion of an internal tag (Flag-6His, named “F”), and preserving the transcriptional activities of both proteins, was developed. This system allowed to demonstrate that Tax-2B is also localized in “nuclear bodies” and that it is able to induce the translocation of the RelA subunit of NF-kB and its recruitment in these structures, as previously observed for Tax-1 (Lamsoul et al., 2005). These innovative results have been recently published (Turci et al., 2009). The intracellular localization of Tax-1 and Tax-2B, which is still a matter of debate (Chevalier et al., 2005; Meertens et al., 2004a; Semmes et al., 1996; Sheehy et al., 2006; Turci et al., 2006), was also compared. The results showed that Tax-2B is more frequently present in the cytoplasm and in “nuclear bodies” as compared to Tax-1. The tagging system devised in this study for Tax-1 and Tax-2B allowed also to compare the transactivating abilities of the two proteins, showing that Tax-1 and Tax-2B share similar, though not identical, capacities to activate gene expression via the NF-kB pathway. In this work, the first time demonstration that Tax-2B is modified by ubiquitination and sumoylation, was also obtained, as has been previously observed for Tax-1 (Lamsoul et al., 2005). The second research line concerned the development of an effective and reproducible system for the detection and quantification of the seven different transcripts of HTLV-2. The genome of this retrovirus is made by two copies of single strand RNA of about 9 Kb that are integrated, as proviral DNA, into the host genome. Several structural and accessory proteins from the full length or the singly or doubly spliced mRNA are produced. Obtaining a precise profile of viral gene expression is considered very useful for understanding the functional role of specific viral genes in the process of viral infection and cellular transformation (Feuer e Green, 2005; Kashanchi e Brady, 2005; Nicot et al., 2005; Li et al., 2009). Therefore, the study was addressed to highlight possible expression differences at different stages of viral infection and in different cellular systems. A TaqMan Real-Time RT-PCR analysis system based on the amplification of cDNA retrotranscripted from total mRNA of HTLV-2 infected cells, was developed. In the first part of the study, three different cell lines were analyzed, namely two T-cell lines, Mo-T e C344 Mo, infected with HTLV-2A and a B-cell line, BJAB-Gu infected with HTLV-2B. The analysis on these cells was performed by sampling them at exponential phase of growth. The data obtained showed notable differences between the two T-cell lines and the B-cell line, since a similar pattern of expression for the two T-cell lines was obtained, whereas the B-cell line presented a different profile. To obtain a more clear-cut information on viral expression patterns, the kinetics of HTLV- 2 transcription in BJAB-Gu and C344 Mo cell lines were analyzed. To synchronize viral expression, the cells were plated at one tenth of optimal growth concentration by ten fold dilution of cells at exponential phase with culture medium. After harvesting cells at different times of division of the two cell lines (24 hours for BJAB-Gu and 48 hours for C344 Mo), the transcripts were analyzed. For BJAB-Gu, an early pattern of expression with a cycle of about 48 hours for tax/rex and p28,p22/p20-1 transcripts followed by a second one at about 96 hours for gag/pol, env and p28,p22/p20-2, was visualized. For C344 Mo, all transcripts showed a peak of expression within the first 24 hours followed by a decrease. Some transcripts (tax/rex and p10/p11) showed a decline up to 48 hours followed by a second peak of expression at 96 hours. For gag/pol and p28,p22/p20rex-2 a decreasing pattern of expression up to 96 hours was observed, with a subsequent peak at 144 hours, followed by a second decline. In conclusion, these cell lines showed a progressive silencing of the gene expression after 144 hours from the initial plating. The experimental work also included the kinetics analysis of expression from PBMCs of an Italian HTLV-2B infected subject. Criopreserved PBMCs were cultured, sampled at 4, 14 and 20 hours and analyzed. For tax/rex transcript, an early and biphasic pattern of expression was observed, while gag/pol and p28,p22/p20-1 and -2 showed a gradual and late profile of expression. Also the env transcript was analyzed, but was below the limit of detection. Altogether these results suggest that the tax/rex transcript is necessary early in infection to transactivate and regulate viral and cellular expression, whereas for other transcripts, which code for structural and accessory proteins, the expression can be delayed. In conclusion, this work has demonstrated that: (i) Tax-1 and Tax-2B shared similar, though not identical, capacity to activate gene expression via NF-kB pathway; (ii) Tax-2B is more frequently present in the cytoplasm and in “nuclear bodies” as compared to Tax-1; (iii) Tax-2B is modified by ubiquitination and sumoylation, It also allowed the identification and quantification of the different HTLV-2 transcripts and showed that the two T-cell lines presented similar patterns of expression, whereas it was different for the B-cell line. The B-cell lines showed a biphasic profile and a progressive decline in the T-cells. In PBMCs obtained from an HTLV-2 infected subject, an early expression of tax/rex was accompanied by a gradual increase of structural transcripts. These results indicate that the infection cycle is based on an earlier transcription of Tax/Rex regulatory proteins followed by that of structural and accessory proteins.