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      Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

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          Summary

          HIV-1 provirus, either as a chromosomal integrant or as an episomal plasmid in HeLa cells, forms a transcription-dependent gene loop structure between the 5′LTR promoter and 3′LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocks 5′ to 3′LTR juxtaposition, indicating that this structure is maintained during transcription. Analysis of mutant or hybrid HIV-1 plasmids demonstrates that replacement of the 5′LTR promoter with CMV or the 3′LTR poly(A) signal with a synthetic element (SPA) permits gene loop formation, suggesting that these interactions are not retroviral specific. In addition, activation of the 5′LTR poly(A) signal or inactivation of the 3′LTR poly(A) signal abolishes gene loop formation. Overall, we demonstrate that both ongoing transcription and pre-mRNA processing are essential for gene loop formation, and predict that these structures represent a defining feature of active gene transcription.

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          Most cited references 40

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          We describe an approach to detect the frequency of interaction between any two genomic loci. Generation of a matrix of interaction frequencies between sites on the same or different chromosomes reveals their relative spatial disposition and provides information about the physical properties of the chromatin fiber. This methodology can be applied to the spatial organization of entire genomes in organisms from bacteria to human. Using the yeast Saccharomyces cerevisiae, we could confirm known qualitative features of chromosome organization within the nucleus and dynamic changes in that organization during meiosis. We also analyzed yeast chromosome III at the G1 stage of the cell cycle. We found that chromatin is highly flexible throughout. Furthermore, functionally distinct AT- and GC-rich domains were found to exhibit different conformations, and a population-average 3D model of chromosome III could be determined. Chromosome III emerges as a contorted ring.
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            Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection.

            The capacity of HIV-1 to establish latent infection of CD4+ T cells may allow viral persistence despite immune responses and antiretroviral therapy. Measurements of infectious virus and viral RNA in plasma and of infectious virus, viral DNA and viral messenger RNA species in infected cells all suggest that HIV-1 replication continues throughout the course of infection. Uncertainty remains over what fraction of CD4+ T cells are infected and whether there are latent reservoirs for the virus. We show here that during the asymptomatic phase of infection there is an extremely low total body load of latently infected resting CD4+ T cells with replication-competent integrated provirus (<10(7) cells). The most prevalent form of HIV-1 DNA in resting and activated CD4+ T cells is a full-length, linear, unintegrated form that is not replication competent. The infection progresses even though at any given time in the lymphoid tissues integrated HIV-1 DNA is present in only a minute fraction of the susceptible populations, including resting and activated CD4+ T cells and macrophages.
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              Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.

              We constructed an infectious molecular clone of acquired immunodeficiency syndrome-associated retrovirus. Upon transfection, this clone directed the production of infectious virus particles in a wide variety of cells in addition to human T4 cells. The progeny, infectious virions, were synthesized in mouse, mink, monkey, and several human non-T cell lines, indicating the absence of any intracellular obstacle to viral RNA or protein production or assembly. During the course of these studies, a human colon carcinoma cell line, exquisitely sensitive to DNA transfection, was identified.
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                Author and article information

                Journal
                Mol Cell
                Molecular Cell
                Cell Press
                1097-2765
                1097-4164
                18 January 2008
                18 January 2008
                : 29
                : 1
                : 56-68
                Affiliations
                [1 ]Sir William Dunn School of Pathology, South Parks Road, University of Oxford, Oxford OX1 3RE, UK
                [2 ]Laboratory of Molecular Medicine, International Center for Genetic Engineering and Biotechnology, Padriciano, 99-34012 Trieste, Italy
                Author notes
                MOLCEL2606
                10.1016/j.molcel.2007.11.030
                2225447
                18206969
                © 2008 ELL & Excerpta Medica.

                This document may be redistributed and reused, subject to certain conditions.

                Categories
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                Molecular biology

                dna

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