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|Autori: ||Orlandi, Chiara|
|Tutor interno: ||ACCOLLA, ROBERTO|
|Titolo: ||Interaction between human oncogenic retrovirus and cellular factors of infected cells.|
|Abstract: ||The purpose of this PhD thesis was to investigate the cellular and molecular interactions between oncogenic retroviruses, particularly HTLV-2 (Human T cell ymphotropic Virus 2), and host factors with potential inhibitory action on retroviral replication. Special attention was concentrated n the host factor CIITA, the HLA class II transactivator.
It was previously shown in this laboratory that CIITA inhibits the transcriptional function of Tax-2 and, consequently, the replication of HTLV-2 virus in human target cells. We tested different fragments of CIITA in 293T cell line, used as a human physiologic system for functional assays with HTLV-2, for the inhibition of Tax-2. We confirm that the Nterminus of CIITA, encompassing the region 1-252, is involved in the block of the Tax-2-mediated transactivation of viral LTR promoter. We also investigated the molecular mechanism at the basis of this effect, and we an unprecedented interaction between CIITA
and Tax-2, that involves the N-term part of CIITA molecule. We identified two
independent regions necessary for the interaction with Tax-2, at the N-terminus and
another one at the C-terminus of CIITA, that are differentially available in the wild type
molecule. We conclude that CIITA could inhibit Tax-2 by directly binding the viral
transactivator, and that this effect may be enhanced by the presence of different factors in common between the host cell and the virus like p300, CBP or PCAF that, together with NF-YB, could help the formation of the inhibiting complex.
We repeatedly observed that when Tax-2 is co-transfected with CIITA, induces a
significant increase of the expression of CIITA, and this effect is evident also with the Nterm deletion mutants and fragments that belong to the central region of CIITA, but not with C-term fragments. We also demonstrate that the rate of CIITA protein increase is dependent on the amount of Tax-2. We identified a region of CIITA that is the target of Tax-2 –mediated increase, including the aminoacids from position 1 to 252.
We asked whether the interaction between the host factor CIITA and the viral transactivator and the increase of CIITA protein could affect also the activity of CIITA on HLA-II promoters. With flow cytometry analysis, we observed that Tax-2 alone does not affect HLA expression, whereas it increases CIITA-mediated expression of HLA-DR on the cell surface. Interestingly, we found also that in the presence of Tax-2 the interaction
between CIITA and NF-YB is significantly more efficient, indicating that CIITA when coexpressed with Tax-2 presents a better functionality on class II genes promoters. We ruled out a possible transcriptional effect of Tax-2 on CMV promoter driving the expression of CIITA, because Tax-2 does not increase the expression of other proteins transcribed from the same CMV promoter, such as GFP and NF-YB, but enhances a CIITA molecule whose expression is under the control of a RSV promoter.
Further studies allowed us to determine that Tax-2 effect is the result of two contributions: Tax-2 stabilizes CIITA mRNA and induces a modification of the kinetic of degradation of CIITA, extending its half-life from 2 to 3 hours.
Together with our previous observations that CIITA inhibits Tax-2 function, these findings
might indicate that HLTV-2 may enhance CIITA protein expression and functionality in
order to exploit its ability to inhibit Tax-2 LTR promoter transactivation and virus
replication. We hypothesize that this “feed back” inhibitory mechanism may control from
one side HTLV virus replication and spreading, but on the other side may also help the virus to maintain a latent state in the infected cells.|
|MIUR : ||MED/09 MEDICINA INTERNA|
|Corso di dottorato: ||Medicina Sperimentale e Oncologia|
|Ciclo di dottorato: ||22|
|Università di conseguimento titolo: ||Università degli Studi dell'Insubria|
|Citazione: ||Orlandi, C.Interaction between human oncogenic retrovirus and cellular factors of infected cells. (Doctoral Thesis, Università degli Studi dell'Insubria, 2010).|
|PhD thesis orlandi completa.doc.pdf||testo completo tesi||1,77 MB||Adobe PDF||non consultabile
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