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      HIV-1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure.

      Cell
      Base Sequence, DNA, Viral, genetics, isolation & purification, Gene Expression, Genes, Viral, HIV, HIV-1, physiology, Humans, Lymphocyte Activation, Molecular Sequence Data, Oligonucleotide Probes, Polymerase Chain Reaction, RNA, Viral, T-Lymphocytes, immunology, microbiology

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          Abstract

          Productive infection of human T lymphocytes by HIV-1 is dependent upon proliferation of the infected cell. Nonproliferating quiescent T cells can be infected by HIV-1 and harbor the virus in an inactive state until subsequent mitogenic stimulation. We use a modification of the polymerase chain reaction method, which is both sensitive and quantitative, to demonstrate that HIV-1 DNA synthesis is initiated in infected quiescent T cells at levels comparable with those of activated T cells. However, unlike that of activated T cells, the viral genome is not completely reverse transcribed in quiescent cells. Although this viral DNA structure can persist in quiescent cells as a latent form, it is labile. We discuss the lability of this HIV-1 DNA structure in relation to a "self-restricting persistent infection" by HIV-1 and propose that this may explain the low percentage of infected cells in the circulation of AIDS patients.

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          Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.

          A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.
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            An inducible transcription factor activates expression of human immunodeficiency virus in T cells.

            Human immunodeficiency virus (HIV) production from latently infected T lymphocytes can be induced with compounds that activate the cells to secrete lymphokines. The elements in the HIV genome which control activation are not known but expression might be regulated through a variety of DNA elements. The cis-acting control elements of the viral genome are enhancer and promoter regions. The virus also encodes trans-acting factors specified by the tat-III and art genes. We have examined whether products specific to activated T cells might stimulate viral transcription by binding to regions on viral DNA. Activation of T cells, which increases HIV expression up to 50-fold, correlated with induction of a DNA binding protein indistinguishable from a recognized transcription factor, called NF-kappa B, with binding sites in the viral enhancer. Mutation of these binding sites abolished inducibility. That NF-kappa B acts in synergy with the viral tat-III gene product to enhance HIV expression in T cells may have implications for the pathogenesis of AIDS (acquired immune deficiency syndrome).
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              A detailed model of reverse transcription and tests of crucial aspects.

              A model of reverse transcription has been devised by which the detailed architecture of ten molecular structures is predicted. The model includes a number of novel features for which experimental evidence is presented. First, growing minus DNA strand is copied from the viral RNA only up to a position about 150 nucleotides from the 5' end of the RNA. Second, plus-strand DNA, after being copied from approximately 600 nucleotides at the 5' end of the minus-strand DNA, then transcribes the first approximately 20 nucleotides of the tRNApro primer (which is covalently attaced to the 5' end of the minus DNA strand). The 3' ends of the minus and plus DNA probably form a hybrid through the homology conferred by the primer binding site sequences. Third, the minus and plus DNA strands are elongated in a continuous fashion resulting in a linear double-stranded DNA molecule containing a 600 nucleotide direct repeat at both ends. The most of the features of the model have experimental support, and it appears to provide a credible description of reverse transcription.
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