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      Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation

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          Abstract

          Background

          HIV-1 replication is critically dependent upon controlled processing of its RNA and the activities provided by its encoded regulatory factors Tat and Rev. A screen of small molecule modulators of RNA processing identified several which inhibited virus gene expression, affecting both relative abundance of specific HIV-1 RNAs and the levels of Tat and Rev proteins.

          Results

          The screen for small molecules modulators of HIV-1 gene expression at the post-transcriptional level identified three (a pyrimidin-7-amine, biphenylcarboxamide, and benzohydrazide, designated 791, 833, and 892, respectively) that not only reduce expression of HIV-1 Gag and Env and alter the accumulation of viral RNAs, but also dramatically decrease Tat and Rev levels. Analyses of viral RNA levels by qRTPCR and RTPCR indicated that the loss of either protein could not be attributed to changes in abundance of the mRNAs encoding these factors. However, addition of the proteasome inhibitor MG132 did result in significant restoration of Tat expression, indicating that the compounds are affecting Tat synthesis and/or degradation. Tests in the context of replicating HIV-1 in PBMCs confirmed that 791 significantly reduced virus replication. Parallel analyses of the effect of the compounds on host gene expression revealed only minor changes in either mRNA abundance or alternative splicing. Subsequent tests suggest that 791 may function by reducing levels of the Tat/Rev chaperone Nap1.

          Conclusions

          The three compounds examined (791, 833, 892), despite their lack of structural similarity, all suppressed HIV-1 gene expression by preventing accumulation of two key HIV-1 regulatory factors, Tat and Rev. These findings demonstrate that selective disruption of HIV-1 gene expression can be achieved.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12977-017-0330-0) contains supplementary material, which is available to authorized users.

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          Most cited references36

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          A highly conserved program of neuronal microexons is misregulated in autistic brains.

          Alternative splicing (AS) generates vast transcriptomic and proteomic complexity. However, which of the myriad of detected AS events provide important biological functions is not well understood. Here, we define the largest program of functionally coordinated, neural-regulated AS described to date in mammals. Relative to all other types of AS within this program, 3-15 nucleotide "microexons" display the most striking evolutionary conservation and switch-like regulation. These microexons modulate the function of interaction domains of proteins involved in neurogenesis. Most neural microexons are regulated by the neuronal-specific splicing factor nSR100/SRRM4, through its binding to adjacent intronic enhancer motifs. Neural microexons are frequently misregulated in the brains of individuals with autism spectrum disorder, and this misregulation is associated with reduced levels of nSR100. The results thus reveal a highly conserved program of dynamic microexon regulation associated with the remodeling of protein-interaction networks during neurogenesis, the misregulation of which is linked to autism.
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            Alternative splicing of human immunodeficiency virus type 1 mRNA modulates viral protein expression, replication, and infectivity.

            Multiple RNA splicing sites exist within human immunodeficiency virus type 1 (HIV-1) genomic RNA, and these sites enable the synthesis of many mRNAs for each of several viral proteins. We evaluated the biological significance of the alternatively spliced mRNA species during productive HIV-1 infections of peripheral blood lymphocytes and human T-cell lines to determine the potential role of alternative RNA splicing in the regulation of HIV-1 replication and infection. First, we used a semiquantitative polymerase chain reaction of cDNAs that were radiolabeled for gel analysis to determine the relative abundance of the diverse array of alternatively spliced HIV-1 mRNAs. The predominant rev, tat, vpr, and env RNAs contained a minimum of noncoding sequence, but the predominant nef mRNAs were incompletely spliced and invariably included noncoding exons. Second, the effect of altered RNA processing was measured following mutagenesis of the major 5' splice donor and several cryptic, constitutive, and competing 3' splice acceptor motifs of HIV-1NL4-3. Mutations that ablated constitutive splice sites led to the activation of new cryptic sites; some of these preserved biological function. Mutations that ablated competing splice acceptor sites caused marked alterations in the pool of virus-derived mRNAs and, in some instances, in virus infectivity and/or the profile of virus proteins. The redundant RNA splicing signals in the HIV-1 genome and alternatively spliced mRNAs provides a mechanism for regulating the relative proportions of HIV-1 proteins and, in some cases, viral infectivity.
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              Human tribbles, a protein family controlling mitogen-activated protein kinase cascades.

              Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.
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                Author and article information

                Contributors
                416 978-2500 , A.balachandran@mail.utoronto.ca
                Rw.wong@mail.utoronto.ca
                stoilov@gmail.com
                sandypan2010@gmail.com
                b.blencowe@utoronto.ca
                pcheung@cfenet.ubc.ca
                prharrigan@cfenet.ubc.ca
                alan.cochrane@utoronto.ca
                Journal
                Retrovirology
                Retrovirology
                Retrovirology
                BioMed Central (London )
                1742-4690
                26 January 2017
                26 January 2017
                2017
                : 14
                : 7
                Affiliations
                [1 ]GRID grid.17063.33, Department of Molecular Genetics, , University of Toronto, ; 1 King’s College Circle, Toronto, ON M5S1A8 Canada
                [2 ]GRID grid.17063.33, Department of Laboratory Medicine and Pathobiology, , University of Toronto, ; Toronto, ON Canada
                [3 ]ISNI 0000 0001 2156 6140, GRID grid.268154.c, Department of Biochemistry, , University of West Virginia, ; Morgantown, WV USA
                [4 ]GRID grid.17063.33, Donnelly Centre, , University of Toronto, ; Toronto, ON Canada
                [5 ]ISNI 0000 0000 8589 2327, GRID grid.416553.0, , British Columbia Centre for Excellence in HIV/AIDS, ; 608-1081 Burrard St., Vancouver, BC Canada
                [6 ]ISNI 0000 0001 2288 9830, GRID grid.17091.3e, Department of Medicine, , University of British Columbia, ; Vancouver, BC Canada
                Article
                330
                10.1186/s12977-017-0330-0
                5267425
                28122580
                0baa97a9-e6ed-40eb-8ae0-70e76a573618
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 28 June 2016
                : 4 January 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000024, Canadian Institutes of Health Research;
                Award ID: HOP-134065
                Award ID: Frederick Banting and Charles Best Canada Graduate Scholarship
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Microbiology & Virology
                hiv-1,rna processing,tat,rev,small molecule inhibitors
                Microbiology & Virology
                hiv-1, rna processing, tat, rev, small molecule inhibitors

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