Date of Award

11-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Sciences

Department

Biochemistry & Molecular Biology

First Advisor

Farah Mustafa

Second Advisor

Tahir A. Rizvi

Abstract

The mouse mammary tumor virus (MMTV) is a prototypic betaretrovirus that causes breast cancer in mice. MMTV regulates its gene expression both at the transcriptional and post transcriptional levels. It has hormonally-regulated promoters controlled by negative regulatory factors in a tissue-specific manner. Recently, we identified a novel 24-nucleotide region at the 5' end of the MMTV genome crucial for MMTV gene expression. Its deletion resulted in disruption of MMTV transcript elongation and stability, leading to a complete loss of Gag gene expression. This conserved element is present within the bifurcated stem loop 4, responsible for gRNA dimerization and encapsidation, into nascent virions. It is called "novel" due to its distinctive location downstream of the major splice donor, but immediately upstream of the Gag ATG, rather than the viral long terminal repeat (LTR), which houses the promoter, transcription factor binding sites, and other regulatory elements that control gene expression. MMTV must transport its full-length genomic RNA (gRNA) post transcriptionally to the cytoplasm without undergoing splicing. This is facilitated by the virally-encoded Rem signal peptide which interacts with the Rem-responsive element (RmRE) to enable the nucleocytoplasmic transport of its gRNA.

Whether the 5’ element is Rem-responsive or has any functional interaction with host/viral factors to facilitate MMTV gene expression has not been elucidated. Here, we examined the role of the 5' element in the transcriptional regulation of MMTV independent of RmRE. Briefly, the 5' element and RmRE were deleted individually or together in a replication-competent molecular clone of MMTV and tested in HEK293T cells treated with the transcription inhibitor Actinomycin D to determine whether the 5’ element could function independent of the RmRE. Quantitative RT-PCRs on RNA isolated from cell lysates immunoprecipitated with anti-RNA polymerase II or anti-pTEFb antibodies revealed a defect primarily in efficiency of transcript initiation and elongation, leading to highly reduced and truncated transcripts in the 5’ element mutants, while nuclear export of the gRNA was primarily affected in the RmRE mutants.

Next, a previously-established reporter gene assay was employed to assess the Rem responsiveness of the 5' element cloned downstream of a luciferase (LUC) gene, flanked by retroviral splice sites. Test of the 5’ element LUC constructs revealed that Rem was unable to rescue splicing of the LUC transcript when it could efficiently rescue it in the presence of RmRE. Newly designed reporter assays were employed to determine the potential of the 5’ element to be transactivated by heterologous viral proteins commonly observed in retroviruses. Test of transactivators from human immunodeficiency virus (HIV) and human T lymphotropic virus (HTLV), Tat and Tax, revealed that the 5’ element could be transactivated by both proteins. Conversely, the MMTV genome could transactivate the HIV-1 TAR element, similar to HTLV-1 Tax. Furthermore, the MMTV genome itself could transactivate the 5’ element in a dose-dependent manner, suggesting that MMTV encodes a viral factor capable of transactivating the 5’ element.

Finally, a series of vectors expressing known viral genes of MMTV were used to identify the factor within the MMTV genome responsible for transactivating the 5’ element. Test of these plasmids suggest that the factor resides within MMTV Pol. Our data further suggests that the viral factor is more similar to HTLV-1 Tax than HIV-1 Tat. Phylogenetic analysis of beta-, delta-, & lentiviruses supports these findings, suggesting that betaretroviruses may be closer to the deltaretroviruses than lentiviruses. Thus, MMTV not only harbors a 5’ cis-acting element similar to HIV-1-TAR/HTLV-1-TRE, but may contain a virally-encoded Tat/Tax-like factor capable of activating MMTV gene expression. Overall, our findings reveal that MMTV is the first betaretrovirus to encode both the Rem/RRE and Tat/TAR- or Tax/TRE-like regulatory systems.

Arabic Abstract


مفهوم جديد حول عملية تكرار فيروس الـMMTV: يمكن لبروتينات الفيروسات القهقرية البشرية HIV-1 TAT و HTLV-1 tax تفعيل العنصر الجديد في المنطقة غير المترجمة ' 5 من جينوم فيروس الـ MMTV

ﻓﯿﺮوس اﻻورام اﻟﺜﺪﯾﯿﺔ (MMTV) ھﻮ ﻓﯿﺮوس ﻧﻤﻮذﺟﻲ ارﺗﺠﺎﻋﻲ ﻣﻦ ﻧﻮع اﻟﺒﯿﺘﺎ ﯾﻌﻤﻞ ﻋﻠﻰ ﻧﻘﻞ اﻟﺤﻤﺾ اﻟﻨﻮوي اﻟﺮﯾﺒﻲ اﻟﺠﯿﻨﻮﻣﻲ ﻛﺎﻣﻞ اﻟﻄﻮل (gRNA) إﻟﻰ اﻟﺴﯿﺘﻮﺑﻼزم، وﻋﻠﻰ ﻏﺮار اﻟﻔﯿﺮوﺳﺎت اﻻرﺗﺠﺎﻋﯿﺔ اﻷﺧﺮى، دون اﻟﺨﻀﻮع ﻟﻠﺘﻘﺴﯿﻢ. ﯾﺘﻢ ﺗﺴﮭﯿﻞ ھﺬه اﻟﻌﻤﻠﯿﺔ ﻣﻦ ﺧﻼل اﻟﺒﺒﺘﯿﺪ اﻟﻔﯿﺮوﺳﻲ اﻟﻤﺸﻔﺮ Rem واﻟﺬي ﯾﺘﻔﺎﻋﻞ ﻣﻊ ﻋﻨﺼﺮ الـ cis، وهو ﻣﺎ ﯾﻌﺮف ﺑﺎﻟﻌﻨﺼﺮ اﻟﻤﺴﺘﺠﯿﺐ ﻟــRmRE) Rem)، ﻟﺘﻤﻜﯿﻦ اﻟﻨﻘﻞ اﻟﺴﯿﺘﻮﺑﻼزﻣﻲ اﻟﻨﻮوي ﻟﻠـgRNA. ﻟﻘﺪ ﺣﺪدﻧﺎ ﻣﺆﺧﺮً ا ﻣﻨﻄﻘﺔ ﺟﺪﯾﺪة ﻣﻜﻮﻧﺔ ﻣﻦ 24 ﻧﯿﻮﻛﻠﯿﻮﺗﯿﺪ، ﺗﻘﻊ ﻓﻲ اﻟﻄﺮف 5’ ﻣﻦ ﺟﯿﻨﻮم الـMMTV، وھﻲ ﻣﻨﻄﻘﺔ ﻣﮭﻤﺔ ﻟﻠﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ اﻟﻔﯿﺮوﺳﻲ. وﻗﺪ أدى ﺣﺬف ھﺬه اﻟﻤﻨﻄﻘﺔ إﻟﻰ اﺿﻄﺮاب ﺷﺪﯾﺪ ﻓﻲ اﺳﺘﻘﺮار ﻧﺴﺨﺔ اﻟﺤﻤﺾ اﻟﻨﻮوي ﻟلـMMTV واﺳﺘﻄﺎلته، ﻣﻤﺎ أدى ﻓﻲ اﻟﻨﮭﺎﯾﺔ إﻟﻰ ﻓﻘﺪان ﻛﺎﻣﻞ ﻟﻠﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ اﻟﮭﯿﻜﻠﻲ ﻟلـ Gag. ﯾﻮﺟﺪ ھﺬا اﻟﻌﻨﺼﺮ ﻓﻲ ﺟﻤﯿﻊ اﻟﺴﻼﻻت اﻟﻤﺨﺘﻠﻔﺔ ﻣﻦ ﻓﯿﺮوس الـMMTV، وهو ﻣﻮﺟﻮد داﺧﻞ اﻟﺤﻠﻘﺔ اﻟﺠﺬﻋﯿﺔ اﻟﻤﺘﺸﻌﺒﺔ اﻟﺮاﺑﻌﺔ (SL4)، وهو ﻣﻜﻮن ﻣﮭﻢ وﻣﺴﺆول ﻋﻤﺎ ﯾﻌﺮف ﺑﺎلـ dimerizationﻟﻠﺤﻤﺾ اﻟﻨﻮوي اﻟﺮﯾﺒﻲ وتغليفه ﻓﻲ ﺟﺰﯾﺌﺎت اﻟﻔﯿﺮوس اﻟﻨﺎﺷﺌﺔ. وﻣﻦ اﻟﺠﺪﯾﺮ ﺑﺎﻟﺬﻛﺮ أنه ﯾُﺸﺎر إﻟﻰ ھﺬا اﻟﻌﻨﺼﺮ ﻋﻠﻰ أنه "ﺟﺪﯾﺪ" ﻧﻈﺮً ا ﻟﻤﻮقعه اﻟﻤﻤﯿﺰ أﺳﻔﻞ الـmajor splice donor، وﻟﻜﻦ ﻣﺒﺎﺷﺮة أﻋﻠﻰ ﻣﻨﻄﻘﺔ الـGag ATG، ﻋﻠﻰ ﻋﻜﺲ اﻟﺘﻜﺮار اﻟﻄﺮﻓﻲ اﻟﻄﻮﯾﻞ اﻟﻔﯿﺮوﺳﻲ (LTR)، اﻟﺬي ﯾﻀﻢ ﺗﻘﻠﯿﺪﯾًﺎ الـpromoter، وﻣﻮاﻗﻊ رﺑﻂ ﻋﺎﻣﻞ اﻟﻨﺴﺦ، واﻟﻌﻨﺎﺻﺮ اﻟﺘﻨﻈﯿﻤﯿﺔ اﻟﻤﻌﺰزة/اﻟﺴﻠﺒﯿﺔ اﻷﺧﺮى اﻟﺘﻲ ﺗﺘﺤﻜﻢ ﻓﻲ اﻟﺘﻌﺒﯿﺮ اﻟﺠﯿﻨﻲ. ﻋﻼوة ﻋﻠﻰ ذﻟﻚ، ﻛﻤﺎ ھﻮ اﻟﺤﺎل ﻣﻊ اﻟﻔﯿﺮوﺳﺎت اﻻرﺗﺠﺎﻋﯿﺔ اﻷﺧﺮى، ﯾﺠﺐ ﻋﻠﻰ ﻓﯿﺮوس الـ MMTV أن ﯾﻘﻮم ﺑﻌﻤﻠﯿﺔ ﻧﻘﻞ اﻟﺤﻤﺾ اﻟﻨﻮوي اﻟﺮﯾﺒﻲ اﻟﺠﯿﻨﻮﻣﻲ ﻛﺎﻣﻞ اﻟﻄﻮل (gRNA) ﺑﺸﻜﻞ ﻛﺎﻣﻞ إﻟﻰ اﻟﺴﯿﺘﻮﺑﻼزم دون اﻟﺨﻀﻮع ﻟﻠﺘﻘﺴﯿﻢ. ﯾﺘﻢ ﺗﺴﮭﯿﻞ ذﻟﻚ ﻣﻦ ﺧﻼل ﺑﺒﺘﯿﺪ الـRem اﻟﻤﺸﻔﺮ ﻓﯿﺮوﺳﯿًﺎ واﻟﺬي ﯾﺘﻔﺎﻋﻞ ﻣﻊ ﻋﻨﺼﺮ الـcis، ﻣﺎ ﯾﻌﺮف ﺑﺎلـ(RmRE)، ﻟﺘﻤﻜﯿﻦ اﻟﻨﻘﻞ اﻟﺴﯿﺘﻮﺑﻼزﻣﻲ اﻟﻨﻮوي ﻟـلـ gRNA اﻟﺨﺎص به.

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