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      Characterization of Tat Antibody Responses in Chinese Individuals Infected with HIV-1

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          Abstract

          HIV-1 Tat is an important regulatory protein involved in AIDS pathogenesis. However, the immunoprofiles of anti-Tat responses remain unclear. We analysed the immunoprofiles of the anti-Tat antibody responses and the neutralizing activities. Out of 326 HIV-1-seropositive individuals, 12.9% were positive for anti-Tat antibodies. We found six different immunological profiles of anti-Tat antibody responses: full-potential response, combined response, N-specific response, C-specific response, full-length Tat-specific response and Tat-related response. These responses represent two types of anti-Tat responses: the major complete response and the alternative C-prone response. A Tat-neutralizing activity is significantly higher in anti-Tat-seropositive samples than anti-Tat-negative or healthy blood-donor samples, and significantly correlates with the anti-Tat reactivities. The data here could contribute to a better understanding of the significance of anti-Tat responses in preventing HIV pathogenesis and could be useful for designing more effective vaccines in the future.

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          Most cited references32

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          A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.

          Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
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            Crystal structure of HIV-1 Tat complexed with human P-TEFb

            Regulation of the expression of the human immunodeficiency virus (HIV) genome is accomplished in large part by controlling transcription elongation. The viral protein Tat hijacks the host cell's RNA polymerase II elongation control machinery through interaction with the positive transcription elongation factor, P-TEFb, and directs the factor to promote productive elongation of HIV mRNA. Here we describe the crystal structure of the Tat•P-TEFb complex containing HIV-1 Tat, human Cdk9, and human Cyclin T1. Tat adopts a structure complementary to the surface of P-TEFb and makes extensive contacts, mainly with the Cyclin T1 subunit of P-TEFb, but also with the T-loop of the Cdk9 subunit. The structure provides a plausible explanation for the tolerance of Tat to sequence variations at certain sites. Importantly, Tat induces significant conformational changes in P-TEFb. This finding lays a foundation for the design of compounds that would specifically inhibit the Tat•P-TEFb complex and block HIV replication.
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              Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

              During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                2 April 2013
                : 8
                : 4
                : e60825
                Affiliations
                [1 ]Department of Microbiology, Second Military Medical University, Shanghai, China
                [2 ]Center for Infectious Diseases, Beijing You’an Hospital, Beijing, China
                [3 ]College of High Altitude Military Medicine, Third Military Medical University, Chongqing, China
                University of South Carolina School of Medicine, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: WP HW. Performed the experiments: QC LL WL YD Huaqun Zhang. Analyzed the data: JC JW. Contributed reagents/materials/analysis tools: LL Hongwei Zhang BS XH TZ HW. Wrote the paper: WL YD.

                Article
                PONE-D-12-25347
                10.1371/journal.pone.0060825
                3614898
                23565278
                a4911b85-98f6-4e4b-9b5c-307cd686dd67
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 21 August 2012
                : 2 March 2013
                Page count
                Pages: 10
                Funding
                This work was funded by the National Natural Science Foundation of China (NSFC) [Grant numbers 30972632 and 30872405], the Shanghai Committee of Science and Technology [08JC1405200], the Chinese National Key Special Project for the Prevention and Control of Major Infectious Diseases [2009ZX10104-105] and the Chinese National Key Special Project for Major New Drug Discovery [2011ZX09506-001]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Virology
                Immunodeficiency Viruses
                Viral Replication
                Pathogenesis
                Mathematics
                Statistics
                Biostatistics
                Medicine
                Anatomy and Physiology
                Immune Physiology
                Antibodies
                Clinical Immunology
                Immunity
                Vaccination
                Vaccines
                Infectious Diseases
                Viral Diseases
                HIV
                Retrovirology and HIV immunopathogenesis

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                Uncategorized

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