Добірка наукової літератури з теми "Oncoproteina Tax"

Transcriptional regulation of the actin-bundling protein and tumor marker Fascin is highly diverse depending on cell and tumor type. Previously, we discovered that the viral oncoprotein Tax-1 of human T-cell leukemia virus type 1 (HTLV-1) considerably enhances Fascin expression in T-cells, depending on classical NF-κB signaling. In this study, we asked if the non-oncogenic Tax-2 of the related HTLV-2 is still able to induce Fascin by using luciferase assays, immunoblot, and qPCR. We found that Tax-2 only slightly induces Fascin expression compared to Tax-1; however, both Tax-1 and Tax-2 comparably activated a 1.6 kb fragment in the human Fascin promoter including Tax-responsive elements. Furthermore, we identified a link between Tax-induced activity of the alternative NF-κB pathway and Fascin induction. While treatment with the second mitochondria-derived activator of caspases (SMAC)-mimetic AZD5582, a compound known to robustly activate alternative NF-κB signaling, did not induce Fascin, combination of AZD5582 with activation of classical NF-κB signaling by Tax-2 significantly induced Fascin expression. In conclusion, our data demonstrate that both classical and alternative NF-κB activity are necessary for strong Fascin induction by the viral Tax oncoproteins, thus, shedding new light on the regulation of Fascin in T-cells and during viral transformation.

AbstractOncogenic transformation of CD4+ T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1–transformed and adult T-cell leukemia/lymphoma patient-derived CD4+ T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1–associated pathogenesis.

Abstract Activation of the NF-κB pathway by the HTLV-I oncoprotein Tax plays a mandatory role in the proliferation and transformation of infected T cells. We have previously demonstrated that Tax is ubiquitylated, mostly on lysine residues located in its carboxy-terminal half. In this study, we investigated the contribution of Tax ubiquitylation on its ability to transactivate the NF-κB pathway. We show that the integrity of C-terminal lysines, and Tax ubiquitylation, are critical for Tax binding to IKK, IKK activation and nuclear translocation of NF-κB. We also report that Tax is post-translationally modified by SUMO on lysine residues also targeted by ubiquitin and that Tax sumoylation occurs in the nucleus and is required for the terminal steps of NF-κB activation. Conversely, Tax ubiquitylation and sumoylation are not involved in CREB activation. Thus, the differential ubiquitylation or sumoylation of the same lysines in Tax regulates essential events controlling the NF-κB pathway, revealing how distinct cellular modifications enrich the functions of this versatile oncoprotein.

AbstractHuman T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide–binding proteins (G proteins) and G protein–coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein β subunit. Interestingly, though the G-protein β subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein β subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1–expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.

ABSTRACT The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G1 phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21CIP. Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.

AbstractThe Really Interesting New Gene (RING) Finger Protein 4 (RNF4) represents a class of ubiquitin ligases that target Small Ubiquitin-like Modifier (SUMO)–modified proteins for ubiquitin modification. To date, the regulatory function of RNF4 appears to be ubiquitin-mediated degradation of sumoylated cellular proteins. In the present study, we show that the Human T-cell Leukemia Virus Type 1 (HTLV-1) oncoprotein Tax is a substrate for RNF4 both in vivo and in vitro. We mapped the RNF4-binding site to a region adjacent to the Tax ubiquitin/SUMO modification sites K280/K284. Interestingly, RNF4 modification of Tax protein results in relocalization of the oncoprotein from the nucleus to the cytoplasm. Overexpression of RNF4, but not the RNF4 RING mutant, resulted in cytoplasmic enrichment of Tax. The RNF4-induced nucleus-to-cytoplasm relocalization was associated with increased NF-κB–mediated and decreased cAMP Response Element-Binding (CREB)–mediated Tax activity. Finally, depletion of RNF4 by RNAi prevented the DNA damage–induced nuclear/cytoplasmic translocation of Tax. These results provide important new insight into STUbL-mediated pathways that regulate the subcellular localization and functional dynamics of viral oncogenes.

ABSTRACT Owing to a single missense mutation in the cell proliferation factor HCF-1, the temperature-sensitive tsBN67 hamster cell line arrests proliferation at nonpermissive temperatures, primarily in a G0/G1 state, and displays temperature-sensitive cytokinesis defects. The HCF-1 mutation in tsBN67 cells also causes a temperature-sensitive dissociation of HCF-1 from chromatin prior to cell proliferation arrest, suggesting that HCF-1-chromatin association is important for mammalian-cell proliferation. Here, we report that the simian virus 40 (SV40) early region, in particular, large T antigen (Tag), and the adenovirus oncoprotein E1A can rescue the tsBN67 cell proliferation defect at nonpermissive temperatures. The SV40 early region rescues the tsBN67 cell proliferation defect without restoring the HCF-1-chromatin association, indicating that these oncoproteins bypass a requirement for HCF-1 function. The SV40 early region also rescues the tsBN67 cytokinesis defect, suggesting that the roles of HCF-1 in cell proliferation and proper cytokinesis are intimately linked. The ability of SV40 Tag and adenovirus E1A to inactivate members of the pRb protein family—pRb, p107, and p130—is important for the bypass of HCF-1 function. These results suggest that HCF-1 regulates mammalian-cell proliferation and cytokinesis, at least in part, by either directly or indirectly opposing pRb family member function.

ABSTRACT Oncogenesis associated with human T-cell leukemia virus (HTLV) infection is directly linked to the virally encoded transcription factor Tax. To activate HTLV-1 transcription Tax interacts with the cellular protein CREB and the pleiotropic coactivators CBP and p300. While extensively studied, the molecular mechanisms of Tax transcription function and coactivator utilization are not fully understood. Previous studies have focused on Tax binding to the KIX domain of CBP, as this was believed to be the key step in recruiting the coactivator to the HTLV-1 promoter. In this study, we identify a carboxy-terminal region of CBP (and p300) that strongly interacts with Tax and mediates Tax transcription function. Through deletion mutagenesis, we identify amino acids 2003 to 2212 of CBP, which we call carboxy-terminal region 2 (CR2), as the minimal region for Tax interaction. Interestingly, this domain corresponds to the steroid receptor coactivator 1 (SRC-1)-interacting domain of CBP. We show that a double point mutant targeted to one of the putative α-helical motifs in this domain significantly compromises the interaction with Tax. We also characterize the region of Tax responsible for interaction with CR2 and show that the previously identified transactivation domain of Tax (amino acids 312 to 319) participates in CR2 binding. This region of Tax corresponds to a consensus amphipathic helix, and single point mutations targeted to amino acids on the face of this helix abolish interaction with CR2 and dramatically reduce Tax transcription function. Finally, we demonstrate that Tax and SRC-1 bind to CR2 in a mutually exclusive fashion. Together, these studies identify a novel Tax-interacting site on CBP/p300 and extend our understanding of the molecular mechanism of Tax transactivation.

I retrovirus umani leucemogeni delle cellule T di tipo 1 e 2 (HTLV-1 e HTLV-2) sono virus oncogeni responsabili di malattie linfoproliferative e neurodegenerative nell’uomo. Un ruolo determinante nel processo di trasformazione cellulare indotto da HTLV è attribuito alla proteina trans-regolatoria Tax, codificata dall’mRNA virale pX. L’oncoproteina Tax di HTLV-1 (Tax-1) è in grado di riprogrammare la progressione dalla fase G1 ad S del ciclo cellulare attraverso molteplici processi molecolari quali il legame diretto di proteine, la repressione o induzione trascrizionale e le modificazioni post-traduzionali. La proteina Tax di HTLV-2 (Tax-2), confrontata con Tax-1, presenta più del 70% di identità aminoacidica; tuttavia dimostra una ridotta attività trasformante rispetto la sua omologa. Questa diversità nella attività patogenetica rende lo studio strutturale e funzionale delle proteine Tax estremamente promettente per la comprensione dei meccanismi cellulari che più specificamente intervengono nella oncogenesi. Tax-1 è una proteina di circa 40 kDa che regola la trascrizione virale attraverso la mediazione di proteine cellulari coinvolte in diversi pathway. Evidenze sperimentali dimostrano che l’attivazione costituiva del signaling NF-kB da parte di Tax-1 è essenziale per la trasformazione e che Tax-1 interagisce con numerosi fattori di questa cascata. Meno note sono le interazioni di Tax-2 con i fattori della stessa via. Scopo della presente ricerca è la definizione di omologie e differenze funzionali tra Tax-2 e Tax-1 nell’attivazione costitutiva del pathway NF-kB. Abbiamo quindi intrapreso uno studio sulle interazioni di Tax-2 con proteine coinvolte nel signaling di NF-kB e sul contributo delle modificazioni post-traduzionali di Tax-1 nell’interazione proteina-proteina. Sono stati scelti tre fattori coinvolti nell’induzione della via canonica NF-kB: il fattore di trascrizione p65/RelA, l’attivatore a monte della via in esame TAB2, e la chinasi IKKe. In questo lavoro di ricerca, per la prima volta si riportano dati che indicano il coinvolgimento del fattore di trascrizione p65/RelA e della proteina TAB2 (TAK1 binding protein 1) nell’attivazione costitutiva della cascata NF-kB indotta dalla proteina virale Tax-2B. Si dimostra, infatti, l’associazione tra Tax-2B e i fattori p65/RelA e TAB2 in saggi di co-immunoprecipitazione in cellule umane e, mediante saggi di luciferasi, si evidenzia l’effetto cooperativo di p65/RelA e/o TAB2 nell’attivazione indotta da Tax-2 dell’espressione da un promotore NF-kB. Il riconoscimento Tax-2/p65 è stato dimostrato inoltre mediante saggi in vitro GST-pull-down. Si dimostra, inoltre, che il riconoscimento di Tax-2 avviene tramite il dominio di TAB2 presente nella regione N-terminale, il quale è necessario per la formazione di un complesso che attivi la cascata NF-kB. Alla luce dei dati qui presentati, si conferma la rilevanza delle interazioni delle proteine Tax con i fattori p65 o TAB2 nell’attivazione della via di segnalazione NF-kB. Lo studio dell’interazione delle proteine Tax con fattori coinvolti nel signaling NF-kB, utilizzando la stessa metodologia, ha individuato una nuova interazione tra le proteine virali Tax-1 e Tax-2B e il fattore IKKe, una chinasi che media la fosforilazione inducibile di p65/RelA e dell’inibitore citoplasmatico IkBa. In considerazione del contributo delle modifiche post-traduzionali di Tax-1 nell’induzione della via NF-kB e nell’associazione con fattori cellulari, è stata testata, in co-immunoprecipitazione, l’abilità di mutanti di Tax-1 di riconoscere p65, TAB2 o IKKe. I risultati di questi esperimenti escludono un ruolo delle modifiche post-traduzionali di Tax-1 nel legame con questi fattori. In conclusione, i dati prodotti in questi studi suggeriscono che Tax-1 e Tax-2B mostrano una capacità simile, ma non identica, di associare e attivare fattori del pathway NF-kB canonico, e che le differenze tra le due proteine virali nella capacità di deregolare le vie di trasduzione del segnale siano da attribuire, almeno parzialmente, alle diversità nell’interazioni con i fattori coinvolti nel pathway NF- kB non-canonico. Successivi studi di associazione con fattori cellulari specificamente coinvolti nella via NF-kB alternativa contribuiranno a chiarire questa ipotesi.
Human T-cell lymphotropic viruses type 1 and type 2 (HTLV-1 and HTLV-2) are related oncoviruses that have been associated with lymphoproliferative and neurodegenerative disorders. The transactivator Tax protein, encoded by the pX region of HTLV genome, is a key factor in cellular transformation. HTLV-1 Tax oncoprotein (Tax-1) reprograms G1 to S progression through multiple mechanistic ways as well as protein-protein binding, transcriptional induction/repression, and post-translational modifications. HTVL-2 Tax (Tax-2) shares more than 70% aa homology whit Tax-1, however Tax-2 has a lower transforming activity than Tax-1. Based on this difference the structural and functional study of Tax proteins can be useful to understand the cellular mechanisms that more specifically take part in oncogenesis. Tax-1 is a 40 KDa transactivator protein which regulates viral transcription by modulating the activity of cellular factors involved in several signal transduction pathways. Tax-1 activation of NF-kB signalling is critical for cellular transformation and while its interaction with NF-kB factors have been intensively investigated little is known about Tax-2 interaction with cellular proteins of this pathway. The aim of this research is the comparison of Tax-2 and Tax-1 for the ability to activate NF-kB pathway. We studied Tax-2 interactions with factors of NF-kB signalling and the contribution of Tax-1 post-translational modifications in the protein-protein interaction. We chose three factors involved in the canonical NF- kB cascade: the transcription factor p65/RelA, the upstream signaling activator TAB2, and the IKKe kinase. In this research, we provide, for the first time, evidence of the involvement of the transcription factor p65/RelA and the protein TAB2 (TAK1 binding protein 1) in Tax-2B-mediated NF-kB activation. In fact, we demonstrate the association of Tax-2B with p65/RelA and TAB2 in co-immunoprecipitation assays in human cells and, by luciferase assays, we highlight the cooperative effect of p65 and/or TAB2 on Tax-2-mediated gene expression activation from NF-kB promoter. Tax- 2/p65 recognition was also shown by in vitro GST-pull-down assays. We also demonstrate that TAB2 is interacting with Tax-2B through a domain that is necessary to form a complex that activates NF-kB cascade. Further analysis of Tax interaction with cellular factors involved in the NF-kB signaling, using the same methodology, identifies a novel interaction between Tax-1 and Tax-2 and IKKe factor, a kinase that mediates inducible phosphorylation of p65/RelA and IkBa. Considering the contribution of Tax-1 post-translational modifications in NF-kB induction and in association with cellular factors, we tested the ability of specific Tax-1 mutants to recognize p65, TAB2 and IKKe by co-immunoprecipitation experiments. The results of this analysis exclude a role of Tax-1 post-translational modifications in the association with these factors. In conclusion, the results obtained in the present study, suggest that Tax-1 and Tax-2B share similar, though not identical, abilities to associate and activate factors of canonical NF-kB pathway. Although we cannot at present explain this diversity, it is tempting to speculate that the differences of the two viral proteins in deregulating signal transduction pathways might be partially attributed to their different capacities to interact with non-canonical NF-kB pathway factors. Comparative studies of Tax-1 and Tax-2 association with cellular factors specifically involved in alternative NF-kB signaling will give new insight to clarify this hypothesis.

Le rétrovirus HTLV-1 qui infecte principalement les lymphocytes T CD4+ est responsable du développement de la leucémie T de l'adulte et d'une pathologie inflammatoire du système nerveux central appelée Paraparésie Spastique Tropicale. Le pouvoir oncogène de ce virus est du à l'expression de deux oncoprotéines virales, les protéines Tax et HBZ. La production de Tax ainsi que de toutes les autres protéines du virus à l'exception d'HBZ est contrôlée par le promoteur sens du virus localisé dans le LTR (Long Terminal Repeat) 5' du virus. La transcription antisens du virus, régulée par le LTR3' donne naissance à deux transcrits l'un épissé, sHBZ, l'autre non, usHBZ. Ces deux ARN une fois traduits permettent la synthèse de deux protéines HBZ différant de quelques acides aminés seulement en N-terminal. Alors que Tax et HBZ provoquent la prolifération accrue des lymphocytes infectés, l'accumulation d'anomalies génétiques, l'immortalisation des lymphocytes, etc.. paradoxalement l'expression de Tax est perdue dans la plupart des cas de leucémie T de l'adulte. Cette perte d'expression passe souvent par la répression de l'expression sens du virus. Ainsi alors que Tax et HBZ participent activement à l'émergence des clones leucémiques, la balance transcriptionnelle est dérégulée en faveur de HBZ au stage final de la transformation par le HTLV-1. Afin de mieux comprendre la pathogenèse associée à HTLV-1 il est donc primordial de comprendre comment la balance entre transcription sens et antisens du virus est régulée dans les premiers temps de l'infection. Alors que l'effet inhibiteur d'HBZ sur la transcription sens est bien décrit, l'effet de la transcription sens et de Tax sur la transcription antisens est encore mal caractérisé. Dans cette étude, nous montrons que dans le contexte de provirus intégrés, le promoteur de sHBZ est moins actif en présence d'une transcription sens. Afin de confirmer ce phénomène, nous avons utilisé deux inhibiteurs pharmacologiques de la transcription sens, la Spironolactone et la Chaetocine pour analyser l'effet d'une diminution du niveau de transcription sens sur la production de sHBZ et usHBZ. Il est montré que l'inhibition de la transcription sens entraine une diminution de la transcription d'usHBZ et une augmentation de la transcription de sHBZ. Les deux transcrits antisens présentent donc une évolution opposée vis à vis de la transcription sens. Pour mieux définir le mécanisme de perturbation de la transcription antisens par la transcription sens, un nouveau modèle a été mis point. Des lignées stables de Jurkat ont été constituée soit avec un plasmide au sein duquel la transcription sens est contrôlée par le CMV, soit ne permettant pas de transcription sens. Ce modèle permettra l'analyse précise des promoteurs du LTR3' et la caractérisation du mécanisme d'inhibition de la transcription de sHBZ par la transcription sens
HTLV-1 retrovirus infects mainly T CD4 lymphocytes and is the causative agent of Adult T cell Leukemia and an inflammatory pathology targeting the central nervous system named Tropical spastic paraparesis. The oncogenic properties of this virus lay in the expression of two oncoproteins, Tax and HBZ. Tax and all viral products except for HBZ are produced from the sense promoter of the virus located in the 5'LTR (Long Terminal Repeat). HTLV-1 antisense transcription leads to the synthesis of two transcripts one spliced, sHBZ, the other one unspliced, usHBZ. These two RNAs once translated give birth to two HBZ protein in which only a few amino acid differ in N-terminal extremity of the proteins. Tax and HBZ induce T lymphocytes proliferation, genomic abnormalities, lymphocytes immortalisation, ... Yet in leukemic cells, most of the time, Tax expression is lost, often by epigenetic repression of the 5'LTR. Thus even if Tax and HBZ actively take part in leukemic clones emergence, HTLV-1 transcriptional balance is deregulated in favor of HBZ at the final stage of transformation. In order to better comprehend how HTLV-1 leads to the development of leukemia, it is essential to understand how HTLV-1 transcriptional balance is regulated in the first steps of HTLV-1 infection. Whereas HBZ inhibitory effect on sense transcription is well described, little is known on the effect of sense transcription on HBZ expression. In this study, it has been shown that sHBZ promoter is less active in HTLV-1 infected lymphocytes with an active sense transcription. To confirm this observation, two pharmacological inhibitors of sense transcription have been caracterized and used to analyze the effect of a change in sense transcription level on sHBZ and usHBZ production. The inhibition of sense transcription is shown to inhibit usHBZ expression and enhance sHBZ transcription. The two antisense transcripts thus exhibit opposite patterns regarding sense transcription. To better define how this repression on sHBZ production is established and how the two promoters in the 3'LTR are regulated, a new model has been built. Jurkat T cells are stably transfected with a plasmid allowing the expression of sense transcripts under the control of the CMV promoter or a plasmid without sense transcription. These models will allow a precise characterization of sHBZ and usHBZ promoter and the deciphering of the inhibition initiated by sense transcription on sHBZ expression

L’attività di ricerca svolta in questa tesi ha riguardato due diversi progetti. Il primo concerne il problema della coinfezione da parte del retrovirus HTLV-2 di soggetti tossicodipendenti italiani che sono infettati da HIV-1, allo scopo di comprendere l’interazione tra i due retrovirus a livello cellulare e stabilire quale possa essere l’effetto sulla progressione dell’AIDS. Il secondo è centrato sullo studio e della funzione delle proteine Tax di HTLV-1 (Tax-1) e HTLV-2 (Tax-2) e sulla caratterizzazione, mediante mutagenesi, dei domini proteici specifici che modulano la funzione stessa. Il primo progetto ha comportato la messa a punto di analisi di PCR quantitativa e la determinazione del carico provirale di HTLV-2 in diversi soggetti infettati e la caratterizzazione molecolare degli isolati di HTLV-2. Dalle analisi compiute sulla popolazione coinfettata e monoinfettata da HIV-1 è risultato che il numero di pazienti “long-term non progressors” verso l’ AIDS è significativamente più alto nel gruppo dei coinfetti HIV-1/HTLV-2 rispetto a quello dei monoinfetti HIV-1. Questi dati indicano chiaramente che HTLV-2 ha un effetto protettivo contro la progressione dell’AIDS. Nel gruppo dei soggetti coinfettati da HIV-1/HTLV-2 sono stati seguiti 5 pazienti sottoposti a terapia antiretrovirale per HIV-1 ed è stato riscontrato un significativo aumento del carico provirale di HTLV-2 e un concomitante decremento in viremia HIV-1 in seguito al trattamento. Questi dati suggeriscono che la terapia antivirale contro l’HIV-1 è inefficace a contenere l’infezione da HTLV-2. La prima parte del secondo progetto ha riguardato l’analisi della localizzazione di Tax-2. A tal fine, il gene tax-2 è stato clonato in un vettore di espressione per cellule di mammifero, aggiungendo in frame all’estremità Cterm la “Green Fluorescent protein” (GFP). L’espressione transiente in cellule HeLa e 293T e la rilevazione tramite microscopia a fluorescenza hanno evidenziato una distribuzione citoplasmatica della proteina in forma punteggiata. Lo stesso procedimento è stato eseguito su mutanti della stessa proteina troncata progressivamente all’estremità C-term e fusa con GFP: 331-GFP, 156- GFP, 115-GFP, 60-GFP. Le immagini al microscopio a fluorescenza hanno confermato una localizzazione di Tax-2 a livello citoplasmatico, ad eccezione del clone 60-GFP, il quale risulta esclusivamente nucleare. L’ulteriore riduzione della proteina ai primi 33 aa mostra una distribuzione della fluorescenza diffusa a tutta la cellula, simile a quella ottenut a dall’espressione della sola proteina GFP. Questi dati confermano la presenza di un “nuclear localization determinant” (NLD) nei primi 60 aa della proteina. Al fine di caratterizzare più precisamente questa parte di Tax-2, sono stati analizzati numerosi mutanti per piccole delezioni aminoacidiche sul clone 60-GFP. Tutti i cloni analizzati, ad eccezione di quello che presenta la delezione 17-20, hanno mostrato una localizzazione nucleare, indicando che mutazioni puntiformi non sono in grado di perturbare la funzionalità del NLD. È stato possibile infine circoscrivere ulteriormente il NLD agli aa 1-41 in quanto, togliendo i primi 21 aa, si riscontra la perdita di funzionalità del NLD stesso. La seconda parte del progetto, riguardante la funzione della proteina Tax, è stata centrata sull’analisi comparativa della localizzazione cellulare di Tax-1 e Tax-2. Per fare questo sono stati clonati i geni tax-1 e tax-2 aggiungendo in frame all’estremità C-term delle proteine dei tag di diverso tipo: la GFP, la Yellow Fluorescent Protein (YFP) (~27 KDa), la proteina Halo (~30 KDa) ed una sequenza aminoacidica di 32 aa contenente l’epitopo V5 (~3 KDa). I cloni di Tax-1, -GFP, -YFP, -Halo e -V5 hanno mostrato una localizzazione prevalentemente nucleare, mentre i cloni di Tax-2, -GFP, -YFP e -Halo sono risultati prevalentemente citoplasmatici. Quando Tax-2 è stata clonata ed espressa con un tag di minor dimensioni quale V5, si è ottenuta una localizzazione prevalentemente nucleare. Questi risultati hanno permesso di dimostrare che l’utilizzo di tag di diverse dimensioni può modulare diversamente la localizzazione delle due proteine, in particolar modo Tax-2. Per identificare le regioni delle due proteine coinvolte nella regolazione della localizzazione cellulare, sono stati fatti esprimere mutanti sia di Tax-1 che di Tax-2 progressivamente troncati all’estremità C-term e fusi con il tag V5. Mettendo a confronto le immagini al microscopio confocale di questi mutanti è stata osservata una distribuzione cellulare molto simile per Tax-1 e Tax-2, anche nelle rispettive forme troncate: prevalentemente nucleari per Tax-1 e Tax-2 “full-length”e per la forma 1-340 e nucleo-citoplasmatiche per gli altri mutanti troncati di dimensioni inferiori. Data la mancanza di un anticorpo specifico contro Tax-2, è necessario di utilizzare sistemi tag per l’espressione ed il riconoscimento di Tax-2 all’interno della cellula per studiarne la localizzazione. Questo ha messo in evidenza che quest’ultima è particolarmente suscettibile alle modificazioni apportate dall’aggiunta di tag in posizione N-term e C-term. Per ovviare a questo problema, si è deciso di preparare costrutti di espressione per Tax-1 e Tax-2 inserendo il tag Flag-H6 all’interno della molecola proteica, tra gli aminoacidi 337 e 338 delle due proteine. Mettendo a confronto la localizzazione di Tax-1 nativa senza tag e quella di Tax-1 utilizzando il tag Flag-H6 in posizione 337, si è visto che questo ultimo non interferisce sui meccanismi che regolano la localizzazione di Tax-1. È stato inoltre possibile stabilire anche per Tax-2 che il tag Flag-H6 posto in posizione 337 permette una corretta lo calizzazione della proteina a livello nucleare. Utilizzando questo sistema di espressione è stato possibile mettere in evidenza alcune caratteristiche funzionali di Tax-2 che non erano ancora state dimostrate. Si è visto infatti che Tax-2 si localizza in modo specifico in strutture nucleari simili ai nuclear bodies (NB) tipici di Tax-1 e che la sua espressione è in grado di determinare un accumulo specifico del fattore RelA di NFKB a livello nucleare nelle stesse strutture. In conclusione questo studio ha permesso di chiarire l’interazione tra HTLV-2 and HIV in soggetti coinfettati ed ha prodotto nuove importanti informazioni sul problema della distribuzione cellulare di Tax-1 e Tax2. Il complesso meccanismo responsabile dell’attività pleiotropica delle due proteine potrà essere chiarito in futuro mediante un'analisi appropriata delle loro modificazioni post-traduzionali
The research activity carried out in this thesis covered two different projects. The first one concerns the problem of the infection by the retrovirus HTLV-2 of Italian intravenous drug users who are also infected by HIV-1, to understand the interaction between the two retroviruses at the cellular level and define the effect of coinfection on AIDS progression. The second is centered on the study and function of Tax proteins of HTLV-1 (Tax-1) and HTLV-2 (Tax-2) and the characterization by mutagenesis of specific protein domains that modulate their function. The first project entailed the development of quantitative PCR analysis and the determination of the proviral load of HTLV-2 in infected subjects and the molecular characterization of HTLV-2 isolates. From the analyses carried out on these subjects it was found that the number of long term non-progressors for AIDS is significantly higher among HIV-1/HTLV-2 coinfected patients than HIV-1 monoinfected cases. These data clearly indicate that HTLV-2 is exerting a protective effect against AIDS progression. Five coinfected subjects undergoing antiretroviral therapy showed a significant increase in HTLV-2 proviral load concomitant to a decrease in HIV-1 viremia, suggesting that the treatment against HIV-1 is ineffective against HTLV-2 infection. The first part of the second project involved the analysis of Tax-2 localization. To this end, tax-2 gene was cloned into an expression vector adding in frame the Green Fluorescent Protein (GFP) to C-term end. The transient expression in HeLa and 293T cells and the detection by fluorescent microscopy revealed that Tax-2 was preferentially distributed in the cytoplasm and formed single dots. To possibly understand the role of C-terminal domain in protein localization, the same expression procedure was performed for Tax-2 mutants progressively truncated at the C-term and merged with GFP: 331-GFP, GFP 156-, 115-GFP, 60-GFP. Fluorescence microscopy confirmed that Tax-2 was localized in the cytoplasm, with the exception of mutant 60-GFP, which was exclusively nuclear. Further reduction to 33 aa fragment showed a widespread cellular fluorescence, similarly that obtained for full length Tax-2-GFP. These data confirmed the presence of a "nuclear localization determinant" (NLD) in the first 60 aa of Tax-2. To more precisely characterize this N-terminal part of the protein, several mutants for single or small amino acid deletions of clone 60- GFP were analyzed. All clones, with the exception of a mutant with 17-20 deletion, presented a nuclear localization, indicating that point mutations are not changing NLD function. Instead, by removing the first 21 aa, NLD function was lost, suggesting that the first 41 aa are necessary for NLD function. The second part of the project on Tax function was centered on the study of comparative cellular localization of Tax-1 and Tax-2. Tax-1 and tax-2 were cloned by adding in frame at C-term different types of protein tags: GFP, Yellow Fluorescent Protein (YFP) (~ 27 KDa ), Halo protein (~ 30 KDa) and a sequence of 32 amino acid containing the epitope V5 (~ 3 KDa). The expression of Tax-1, -GFP, -YFP, -Halo and -V5 showed a predominantly nuclear localization, whereas that of Tax-2 linked to GFP, YFP or Halo was predominantly cytoplasmic. When Tax-2 was cloned and expressed using the small tag V5, a predominantly nuclear localization was obtained. These results demonstrated that using tags of different sizes can induce different localizations of the two proteins. To identify the regions of the two proteins involved in regulating cellular localization, Tax-1 and Tax-2 mutants progressively truncated at the C-term and merged with the tag V5 were constructed and expressed. A very similar cellular distribution for Tax-1 and Tax-2 was obtained, as visualized by confocal microscopy: predominantly nuclear for both Tax-1 and Tax-2 full length and 1- 340 forms and cytoplasmic for truncated mutants below the first 300 aa. Since no adequate antibody is available to recognise native Tax-2 expression inside the cell, adding a tag to the terminal parts of the protein becomes necessary, though these conformational changes could result in abnormal localization effect. It was thus decided to prepare constructs for Tax -1 and Tax-2 expression by inserting internally to the protein structure the tag Flag- H6 between amino acids 337 and 338. By comparing the localization of native Tax-1 without tags and Tax-1 with the 337 Flag-H6, it was found that this does not interfere with the mechanisms regulating Tax-1 localization and function. It was also found that 337 Flag-H6 presented a nuclear localization and was adequately functional. Using this expression system allowed to show that also Tax-2 is located in specific nuclear complexes that are similar, but not identical, to the nuclear bodies (NB) formed by Tax-1 and that their expression is responsible for a specific accumulation of RelA NFKB factor at nuclear level. In conclusion, this study has allowed further understanding of the crossinteraction between HTLV-2 and HIV in coinfected subjects, and has given new important clues on the problem of cellular distribution and function of Tax-1 and Tax-2. The complex mechanisms that are responsible for the pleiotropic activities of the two proteins will be further clarified in the future by investigating their post-translational modifications.