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Kinetics and regulation of HTLV-1 gene expression
Full text linkABSTRACT
Human T-Lymphotropic virus type 1 (HTLV-1) is the causative agent of two distinct pathologies, adult T-cell leukemia/lymphoma (ATLL), an aggressive malignancy of mature CD4+ T-cells, and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM), a demyelinating neurodegenerative disease.
The HTLV-1 expression strategy is characterized by the production of plus- and minus-strand transcripts, alternative splicing and polycistronic translation. This strategy greatly increases the coding potential of the virus, resulting in expression of several regulatory and accessory genes (Tax, Rex, p12, p13, p21rex, p30tof and HBZ) in addition to the structural proteins and virion-associated enzymes common to all retroviruses (Gag, Pro, Pol and Env).
In spite of over 30 years of studies, several key features of the HTLV-1 life cycle and pathogenicity remain obscure. In particular, it is still unclear whether HTLV-1 gene expression is characterized by latency patterns, whether the different viral genes follow distinct kinetics of expression and, if this is the case, which molecular mechanisms control these phenomena.
The work described in the present thesis was aimed at understanding these aspects of HTLV-1 regulation. To this end we optimized a Real Time RT-PCR method using splice-site-specific primers to quantitate the different HTLV-1 transcripts and their kinetics of expression in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1-infected individuals and in cells transfected with HTLV-1 molecular clones. Results indicated that expression of HTLV-1 mRNAs follows a distinct timing upon reactivation of viral expression, with the mRNA coding for the Tax and Rex regulatory proteins acting as an early "master" transcript preceding expression of the other viral transcripts.
Although it is commonly accepted that Rex acts at a post-transcriptional level controlling the nuclear export and stability of viral mRNAs coding for the virion-associated proteins, the Rex-dependency of tax/rex, p12, p13, p21rex, p30tof and hbz transcripts has not been investigated so far. To test if the kinetics of HTLV-1 gene expression might be dependent on Rex function and to determine the Rex-dependence of individual HTLV-1 mRNAs, we generated a Rex knock-out HTLV-1 molecular clone and analyzed the nucleo-cytoplasmic compartmentalization of the viral mRNAs. Results demonstrated the strict Rex-dependency of the “two-phase” kinetics and revealed strong nuclear retention of hbz mRNAs, supporting their function as non-coding transcripts. Furthermore our results revealed that the Rex-responsiveness of the different HTLV-1 mRNAs is determined by a novel 72-nucleotides cis-acting regulatory sequence located upstream of exon 3.
Mathematical modelling underscored the importance of a temporal delay between the Tax and Rex functions, which was supported by experimental evidence of a delayed accumulation and longer half-life of Rex compared to Tax.
These data provide evidence for a temporal pattern of HTLV-1 gene expression, reveal major differences in the intracellular compartmentalization of HTLV-1 transcripts and, importantly, provide clues to a long-standing paradox of HTLV-1 regulation, i.e. the different Rex-dependence of viral transcripts in spite of the presence of the Rex-responsive element (RxRE) in the 3' untranslated region of all viral mRNAs.RIASSUNTO
Il virus T-linfotropico umano di tipo 1 (HTLV-1) è l’agente eziologico di due distinte patologie, la leucemia/linfoma a cellule T dell’adulto (ATLL, adult T-cell leukemia/lymphoma), un'aggressiva neoplasia a carico dei linfociti T CD4+ maturi, e della paraparesi spastica tropicale/mielopatia associata ad HTLV-1 (TSP/HAM, tropical spastic paraparesis/HTLV-1-associated myelopathy), una patologia degenerativa del sistema nervoso centrale.
La strategia di espressione genica di HTLV-1, caratterizzata dalla produzione di trascritti a partire da promotori localizzati sia nel filamento positivo che in quello negativo del genoma virale, da splicing alternativo e da traduzione bicistronica, incrementa notevolmente la capacità codificante di HTLV-1, con la conseguente espressione di numerosi geni regolatori ed accessori (Tax, Rex, p12, p13, p21rex, p30tof e HBZ) in aggiunta alle proteine strutturali e agli enzimi associati al virione, comuni a tutti i retrovirus (Gag, Pro, Pol ed Env).
Nonostante oltre 30 anni di studi, diversi aspetti chiave del ciclo vitale di HTLV-1 e della sua patogenicità rimangono tutt'oggi non noti. In particolare, non è ancora chiaro se l'espressione genica di HTLV-1 sia caratterizzata da stadi di latenza, se i diversi geni virali presentino cinetiche di espressione distinte e quali meccanismi molecolari possano controllare questi fenomeni.
Gli studi descritti nella presente tesi sono stati mirati a comprendere questi aspetti della regolazione genica di HTLV-1. A questo scopo abbiamo sviluppato un protocollo di Real Time RT-PCR associato all'impiego di primer specifici per le diverse giunzioni di splicing al fine di quantificare i diversi trascritti codificati da HTLV-1 e di analizzarne le cinetiche di espressione sia in cellule mononucleate di sangue periferico isolate da individui infettati con HTLV-1, che in cellule trasfettate con cloni molecolari di HTLV-1. I risultati ottenuti indicano che l'espressione degli mRNA codificati da HTLV-1 segue una precisa cinetica dopo riattivazione dell'espressione virale: l'mRNA codificante le proteine regolatrici Tax e Rex agisce come trascritto precoce che precede l'espressione degli altri geni virali.
Sebbene sia comunemente accettato che Rex eserciti la sua funzione a livello post-trascrizionale controllando l'esporto nucleare e la stabilità degli mRNA che codificano le proteine associate al virione, fino ad oggi non è mai stata investigata la Rex-dipendenza dei trascritti p12, p13, p21rex, p30tof e hbz. Al fine di testare se le cinetiche di espressione genica di HTLV-1 osservate potessero dipendere dalla funzione di Rex e al fine di determinare la Rex-dipendenza dei singoli mRNA virali, abbiamo generato un clone molecolare di HTLV-1 knock-out per Rex e analizzato la compartimentalizzazione nucleo-citoplasmatica dei trascritti virali. I risultati ottenuti hanno dimostrato la stretta Rex-dipendenza delle cinetiche di espressione a "due fasi" ed hanno rivelato una forte ritenzione nucleare degli mRNA codificanti HBZ, supportando la loro funzione come trascritti non codificanti. Inoltre, i risultati ottenuti hanno dimostrato che la responsività a Rex dei differenti mRNA virali potrebbe essere determinata dalla presenza di una sequenza regolatoria di 72 nucleotidi che agisce in cis, localizzata a monte dell'esone 3.
Infine, analisi matematiche hanno sottolineato l'importanza di un ritardo temporale tra le funzioni di Tax e di Rex, supportata dall'evidenza sperimentale di un ritardo nell'accumulo e di un'emivita più prolungata di Rex rispetto a Tax.
I dati ottenuti in questo studio forniscono l'evidenza di una regolazione temporale dell'espressione genica di HTLV-1, rivelano una differente compartimentalizzazione degli mRNA virali e offrono una possibile spiegazione di un paradosso ancora irrisolto della regolazione di HTLV-1, ovvero la differente Rex-dipendenza dei trascritti virali, nonostante la presenza della sequenza responsiva a Rex (RxRE, Rex-responsive element) nella regione 3' non tradotta di tutti i trascritti virali
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).
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