Download or read book Human T Cell Leukemia Virus Type 1 Tax Gene Transduction Into Primary Cells Using Lentivirus Vectors written by Stephan H. Wrzesinski and published by . This book was released on 2000 with total page 232 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Molecular Analysis of Human T cell Leukemia Virus Type 2 Accessory Protein written by Brenda Michiyo Yamamoto and published by . This book was released on 2009 with total page 162 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: The human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are two pathogenic retroviruses. Although both viruses share a common genome organization and amino acid homology in common viral proteins, the incidence of disease with infection is distinct. Infection with HTLV-1 may result in the development of adult T-cell leukemia/lymphoma (ATL), an aggressive neoplastic disease, or a variety of immune-mediated/inflammatory disorders such as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), whereas HTLV-2 is less pathogenic. Our studies focused on the open reading frame II encoded p28 protein of HTLV-2, which has been shown to negatively regulate viral expression by the nuclear retention of the tax/rex mRNA. A similar post-transcriptional regulatory function has been observed with HTLV-1 ORF-II p30. However, p28 contrasts p30 in that there appears to be no significant transcriptional effects. In Chapter 2, we examined the functional significance of p28 in HTLV-2 infection, proliferation, and immortalization of primary T-cells in culture, and viral infection and survival in a rabbit model of HTLV infection. We generated a novel HTLV-2 p28 termination clone (HTLV2Deltap28) in which a stop codon had been introduced into the p28 sequence without altering the amino acid sequence of the overlapping regulatory proteins, Tax and Rex. In short-term proliferation and long-term immortalization coculture assays, HTLV2Deltap28 infected and immortalized primary human T-cells, similar to wtHTLV-2. However, HTLV2Deltap28 had a lower capacity to establish persistent infection in rabbits, indicating the in vivo importance of HTLV-2 p28. These results are consistent with the hypothesis that p28 repression of Tax and Rex-mediated viral gene expression allows infected cells to avoid immune recognition and elimination, or acts to enhance early viral spread by enhancing the survival of HTLV-2 infected cells. In Chapter 3, we generated and characterized various dual-promoter and single-promoter lentiviral expression vectors. Post-transduction, p28 protein was readily detected with the dual-promoter vectors in 293T cells but not in Jurkat T-cells. The differential p28 protein expression was found to be due to cell-type specific translation mechanisms. To circumvent this problem we utilized a single-promoter lentiviral vector that expresses p28 via the murine stem cell virus (MSCV)-promoter, which resulted in efficient p28 protein expression in both T-cell lines and primary human CD8+ T-lymphocytes. In Chapter 4, the capacity of p28 to modify cellular gene expression was examined. In transient transfection studies, low doses of p28 modulated CRE- and NF[kappa]B-driven reporter constructs in 293T cells, suggesting the ability of p28 in modulating cellular gene expression. Interestingly, transduction of Jurkat T-cells with the lentiviral p28 expression vector had no significant effect on cellular proliferation. Additionally, initial analysis of global cellular gene expression by microarray analysis suggests that p28 results in nominal alterations in cellular gene expression. Collectively, data presented in this thesis indicates that p28 is critical for the establishment and survival of HTLV-2, compatible with the conclusion that the regulation of HTLV gene expression is a tightly controlled and complex process. Ultimately, while minimal, the impact of p28 upon cellular genes likely contributes to HTLV-2 establishment of infection in vivo.

Download or read book Lentivirus Gene Engineering Protocols written by Maurizio Federico and published by Springer Science & Business Media. This book was released on 2008-02-03 with total page 310 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cell gene engineering is emerging as a field with outstanding impact, not only in medicine/biology, but also, and perhaps most importantly, in agriculture and in all those food sciences involved in the fight against world hunger. Lentivirus vector-based technologies represent the last frontier in the development of powerful and reliable methods for both in vitro and in vivo gene transfer in eukaryotic animal cells. Although the design of lentivirus vectors is closely reminiscent of those already successfully applied to the construction of oncoretroviral vectors, some unique features, e.g., the ef- ciency in transducing both postmitotic and stem cells, render the use of lentivirus vectors invaluable. It has been a great pleasure to edit Lentivirus Gene Engineering Pro- cols, owing in part to the high level of enthusiasm that the authors dem- strated in contributing to this book. The fact that so many outstanding scientists engaged in lentivirus vector research have provided articles renders it so- thing more than a technical handbook. In addition to detailed descriptions of the most innovative methodologies, the reader may find very informative ov- views concerning both theoretical and practical aspects of the origin and the development of diverse lentivirus vector types. This, in my opinion, rep- sents a unique added value of this volume, which should help our work resist the passage of time, to which books such as this are particularly sensitive.

Download or read book Transformation Studies of Human T cell Leukemia Virus with Emphasis on the Role of Tax and Rex written by Jianxin Je and published by . This book was released on 2003 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: HTLV-1 and HTLV-2 both transform human primary T cells but the precise transformation mechanism remains to be elucidated. We studied two HTLV regulatory proteins, Tax and Rex, and their role in HTLV-mediated cellular transformation. HTLV-1 has a preferential transformation tropism of CD4+ T cells, whereas HTLV-2 transforms primarily CD8+ T cells. Since Tax is critical for cellular transformation and differences have been identified between Tax-1 and Tax-2, we hypothesize that the viral determinant of transformation tropism is encoded by Tax. Using molecular clones of HTLV-1 (Ach) and HTLV-2 (pH6neo) we constructed recombinants in which Tax and overlapping Rex genes of the two viruses were exchanged. p19 Gag expression from proviral clones transfected into 293T cells indicated that both recombinants contained functional Tax and Rex but with significantly altered activity as compared to the wild-type clones. Stable transfectants expressing recombinant viruses were established, irradiated, and cocultured with peripheral blood mononuclear cells (PBMC). Both recombinants were competent to transform T-lymphocytes with efficiency similar to the parental viruses. Flow cytometry analysis indicated that HTLV-1 and HTLV-1/TR2 had a preferential tropism for CD4+ T cells and HTLV-2 and HTLV-2/TR1 had a preferential tropism for CD8+ T cells. Our results indicate that tax/rex in different genetic backgrounds display altered functional activity but ultimately do not contribute to the different in vitro transformation tropism. We also studied the contribution of Rex in HTLV-1-mediated immortalization of primary T-cells in vitro and viral survival in a rabbit animal model. Our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo. Efficient HTLV replication requires Rex/RxRE regulation of incompletely spliced viral mRNAs that encode the viral enzymatic and structural proteins. Overall, our results indicate that post-transcriptional control elements identified in other viruses have a partial capacity to substitute for HTLV Rex/RxRE function, although the low activities of these elements are insufficient to maintain viral replication and virus spread in culture. Together this work provides important information on the role of Tax and Rex on HTLV replication and cellular transformation and further insight into the biological differences between HTLV-1 and HTLV-2.

Download or read book Lentiviral Vectors and Gene Therapy written by David Escors and published by Springer Science & Business Media. This book was released on 2012-03-23 with total page 110 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene therapy was conceived during the early and mid part of the 20th century. At first, it was considered a revolutionary biomedical procedure, which could potentially cure any disease for which the molecular bases were understood. Since then, gene therapy has gone through many stages and has evolved from a nearly unrealistic perspective to a real life application. Clinical efficacy in humans was demonstrated at the beginning of this century after its successful application in small-scale clinical trials to cure severe immunodeficiency in children. However, their successes were overshadowed some time later by the occurrence of vector-related leukaemia in a number of treated children. It is in this context that lentiviral vectors have appeared, with improved efficiency and, possibly, increased biosafety. Very recently, the first clinical trials with lentivectors have been carried out with some success. This Brief firstly defines gene therapy, and places lentivectors within this fascinating therapeutic strategy. Then follows a comprehensive description of the development of retroviral and lentiviral vectors and how to specifically target distinct cell types and tissues. The authors also discuss the application of lentivector gene therapy for the treatment of cancer and autoimmune diseases, ending with the application of lentivectors in human gene therapy clinical trials.

Download or read book Molecular Analysis of Human T cell Leukemia Virus Regulatory and Accessory Proteins written by Ihab H. Younis and published by . This book was released on 2005 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are closely related pathogenic human retroviruses. Although, they both transform human primary T cells in vitro, in humans, HTLV-1 is the causative agent for ATLL and HAM/TSP, whereas HTLV-2 disease association is less clear. In this dissertation, we report molecular studies regarding the regulation of HTLV replication and its impact on viral persistence in vivo. In Chapter 2, we generate a novel HTLV-1 clone (H1IT) in which the two regulatory proteins, Tax and Rex, have been separated in an attempt to provide a better reagent to study mutants of these proteins in the context of the provirus and analyze their contribution to HTLV-mediated transformation. In vitro data indicates that H1IT is replication competent and is capable of cellular transformation of primary human T-cells. However, H1IT was unable to persist in vivo, emphasizing the importance of temporal and quantitative regulation of Tax RNA to viral replication. In Chapter 3, we report that both HTLV-1 and HTLV-2 have evolved accessory genes whose products are able to restrict viral replication at a post-transcriptional level. The HTLV-1 p30 and the related HTLV-2 p28 proteins inhibit both Tax and Rex by binding to and retaining tax/rex mRNA in the nucleus, thereby inhibiting virion production. In Chapter 4, we show that p28 is recruited to the viral promoter in a Tax-dependent manner. After recruitment to the promoter, p28 or p30 travels with the transcription elongation machinery until its target mRNA is synthesized. Since the above data is consistent with a critical role of these accessory proteins in viral persistence in vivo, in Chapter 5, we used an animal model of HTLV infection to study the specific contribution of p28 on HTLV-2 survival. In this study, all wtHTLV-2 infected rabbits showed persistent infection, whereas those infected with HTLV-2[delta]p28 were able to eliminate the virus as early as 2 weeks, indicating that p28 is critical for early viral infectivity, spread and/or persistence in rabbits. Collectively, data presented within this dissertation support the conclusion that the regulation of HTLV gene expression a complicated but a tightly controlled process.

Download or read book Human T cell leukemia virus 1 HTLV 1 infection associated pathology and response of the host written by Roberto S. Accolla and published by Frontiers Media SA. This book was released on 2023-06-30 with total page 248 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Regulation of Human T cell Leukemia Virus Type 1 Infection and Replication written by Gunjan Choudhary and published by . This book was released on 2010 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt: Retroviruses have evolved complex mechanisms to regulate their cellular tropism and gene expression. It is generally accepted that productive infections proceed via interactions between viral envelope molecules and specific receptors on the host cell surface. Currently, there is no known receptor for HTLV-1, though a number of factors that enhance entry have been identified. In an effort to identify a cellular receptor or attachment factor for HTLV-1, we carried out a retroviral cDNA library screen, in which cDNA from permissive HeLa S3 cells was introduced into poorly susceptible NIH 3T3 cells. These cells were selected after infection with HTLV-1 envelope pseudotyped viral particles expressing a drug resistance gene. We isolated approximately 460 cDNAs, of which 20 were prioritized as potential candidates. These candidates are being tested to determine if they participate in viral entry. In addition to encoding the structural and enzymatic genes common to all retroviruses, HTLV-1 also encodes several accessory genes which contribute to viral replication and the maintenance of gene expression. A newly identified viral gene, HTLV-1 bZIP factor or hbz, has been shown to have pleiotropic effects as it functions differently in its protein and mRNA forms. In an effort to elucidate its role in HTLV-1 replication, we identified a novel function. Mutations that abrogated the hbz mRNA or disrupted a stem-loop in hbz mRNA, or mutations that eliminated or truncated the HBZ protein were introduced in a functional molecular clone of HTLV-1. The protein and stem-loop mutants had no effect on viral gene expression. However, the mutant that disrupted hbz mRNA expressed lower levels of tax mRNA, suggesting that hbz promotes tax expression. We found that this effect of hbz was indirect, as hbz represses another accessory gene, p30II, which is known to sequester tax mRNA in the nucleus. These results provide new insights into the regulation of HTLV-1 infection, specifically viral entry and gene expression.

Download or read book Lentiviral Vectors written by Didier Trono and published by Springer Science & Business Media. This book was released on 2012-12-06 with total page 261 pages. Available in PDF, EPUB and Kindle. Book excerpt: For the first time a compilation of chapters that depict the biological bases underlying the development of lentiviral vectors, the techniques involved in the manufacture of this new gene delivery tool, and its most promising applications.

Download or read book Subcellular Localization of Human T cell Leukemia Virus Type 1 Tax Oncoprotein written by Kimberly Anne Fryrear and published by . This book was released on 2008 with total page 218 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Kinetic Analysis of Human T cell Leukemia Virus Type 1 Gene Expression written by Min Li and published by . This book was released on 2008 with total page 170 pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) are closely related human retroviruses that transform T lymphocytes in cell culture and persist in infected individuals. HTLV-1 infection is clearly associated with leukemia/lymphoma and neurological disease, whereas HTLV-2 disease association is less compelling. HTLV replication and survival requires the expression of multiple gene products from an unspliced and a series of highly related alternatively spliced mRNA species. To date, the levels of viral gene expression throughout the process of infection, cellular transformation, and pathogenesis have not been experimentally assessed. We posit that having a concise viral gene expression profile will provide important insight into the function of specific viral genes and their role in the biology and pathogenesis of HTLV-1.

Download or read book Transcriptional Control of Human T cell Leukemia Virus Type 1 in Primary Lymphocytes written by Garret C. Newbound and published by . This book was released on 1997 with total page 338 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Human T cell Leukemia Virus written by Peter K. Vogt and published by Springer. This book was released on 1985 with total page 284 pages. Available in PDF, EPUB and Kindle. Book excerpt: Characteristic features in common with the genome of other retroviruses: long terminal repeats (L TR), and coding regions for internal proteins (gag), for re verse transcriptase (pol), and for glycosylated virion surface proteins (env) , ar ranged in the sequence gag, pol, env from the 5' to the 3' end of the genome. However, the HTL V genome also contains some specific features not shared with all other retroviruses: the LTR regions are unusually long (745 base pairs, with 298 base pairs constituting the R region), but unlike the long L TRs of mouse mammary tumor viruses, they do not contain open reading frames. A stretch of noncoding sequences separates the gag and the pol genes. Most interestingly, the HTLV genome contains a region between the 3' end of the env gene and the L TR, called the pX region, that encompasses four open reading frames. Leukemic T cells freshly obtained from patients contain the HTL V provirus but usually do not express it. However, once established in culture, these cells produce viral proteins and release type C particles. Likewise, T cells infected and transformed by HTL V in vitro synthesize virus. Such producing cell lines have been widely used in seroepidemiological surveys and continue to be of importance for detailed studies of viral proteins and nucleic acids.

Download or read book Role of Human T lymphotropic Virus Type 1 P30 II and Surface Envelope as Determinants of in Vivo Pathogenesis written by Lee Silverman and published by . This book was released on 2005 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Human T-cell leukemia virus type 1 (HTVL-1) is the first identified human retrovirus. In addition to gag, pol, and env genes, HTLV-1 contains four open reading frames (ORF) within its pX region. ORF II encodes p30II and p13II. Herein, we determine the in vivo significance of p30II by inoculating rabbits with cell lines expressing either a wild-type clone of HTLV-1 (ACH. 1) or a p30II mutant clone (ACH. 30.1). Compared to ACH. 1 rabbits, ACH. 30.1 rabbits had lower antibody titers, a smaller percentage of seropositive animals, a smaller percentage of animals with provirus in peripheral blood mononuclear cells (PBMC), and lower proviral loads. Sequencing revealed that provirus in ACH. 30.1 provirus positive rabbits had reverted to wild-type sequence. We conclude that there is strong selective pressure for expression of p30II in vivo. We next sought to determine if p30II modulates cellular apoptosis. A greater percentage of ACH. 30.1 cells were induced into apoptosis compared to ACH. 1 cells following treatment with camptothecin (specific for S-phase of cell cycle). There was no difference in apoptosis induction between ACH. 30.1 and ACH. 1 cells following treatment with etoposide (intrinsic pathway) or TRAIL (extrinsic pathway). p30II did not modulate susceptibility to apoptosis when expressed in 293T cells or in Jurkat T cells. Expression of p30II in Jurkat T cells reduced cell proliferation by delaying onset of division. Although p30II does not modulate susceptibility to apoptosis, it does reduce cell proliferation. HTLV-1 Env Ser75Ile, Asn95Asp, and Asn195Asp mutants are able to immortalize lymphocytes in vitro. Herein, we examine the effects of these mutations in rabbits via inoculations with ACH. 75, ACH. 95, ACH. 195, and ACH. 1 cell lines. All mutations were maintained in vivo. ACH. 75 and ACH. 95 rabbits had decreased antibody responses to Gag and Env. One ACH. 195 rabbit had an antibody response to HTLV-1 proteins and HTLV-2 Env. Another ACH. 195 rabbit had provirus in PBMC but no serologic response. ACH. 195 rabbits had a diminished antibody response to Env surface (SU) protein. PBMC proviral loads did not correlate with antibody response to SU. These mutations in HTLV-1 Env SU alter proviral loads and antibody responses against Env but do not prevent virus replication in vivo.

Download or read book Studies with the Human T cell Leukemia Virus Tax and Rex Positive Trans regulatory Proteins written by Matthew David Anderson and published by . This book was released on 2004 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Abstract: Retroviruses have highly organized genomes that encode a large number of proteins from a relatively short nucleic acid sequence. They utilize a variety of methods to achieve this, including the use of overlapping reading frames and the production of multiple RNAs through alternative splicing. In order to efficiently express RNAs encoding structural and enzymatic proteins, complex retroviruses like human T-cell leukemia virus (HTLV) must first express a doubly spliced RNA encoding both the Tax and Rex positive transregulatory proteins in separate but partially overlapping reading frames. As a result, functional studies of each of these proteins in the context of replicating virus, particularly mutational studies, present unique challenges. This dissertation both describes and utilizes experimental systems for the study of Tax and Rex that model in vivo in the context of replicating virus in human T cells, the natural target of HTLV infection. As a whole, the work described in this dissertation uses methods for the study of the HTLV regulatory proteins Tax-1 and Rex-2 that closely approximate conditions that occur in in vivo HTLV infections. Ideally this is an infection of primary human T-lymphocytes. This approach provides highly relevant insight into the molecular mechanisms utilized by these proteins, validates previous results obtained from in vitro studies as well as extends and refines our understanding of the way these proteins function in natural infection.

Download or read book Role of the Tax Gene of the Human T cell Leukemia Virus Type 2 in Transformation of Human T lymphocytes written by Ted Milburn Ross and published by . This book was released on 1996 with total page 252 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book The Human T cell Leukemia Virus Type 1 Tax Transactivation of Viral and Cellular CRE Elements written by Laurie Mandy Connor and published by . This book was released on 1997 with total page 286 pages. Available in PDF, EPUB and Kindle. Book excerpt: