This project focuses on HIV-1 replication in cultured cells to develop understandings of viral pathogenesis and the cellular factors that influence viral gene expression. Examples of important scientific advances that we have achieved in the 2011 to 2012 period are outlined below. RNA helicases are ubiquitous in plants and animals and function in many cellular processes. Retroviruses, such as human immunodeficiency virus (HIV-1), encode no RNA helicases in their genomes and utilize host cellular RNA helicases at various stages of their life cycle. We have continued our study of RNA helicases in HIV-1 replication. We have identified several novel RNA helicases that activate HIV-1 post-transcriptional gene expression and an RNA helicase that represses HIV-1 replication through the interferon pathway. HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q(35)VCF(38), which resembles the PP1-binding """"""""RVxF"""""""" motif present on PP1-binding regulatory subunits. We have found that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. We have identified small molecule compounds that target the """"""""RVxF""""""""-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. Our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication. Our work is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA. In FY 2013, we published our findings that NEAT1 long noncoding RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression. Most of the human genome is transcribed into protein-noncoding RNAs (ncRNAs), including small ncRNAs and long ncRNAs (lncRNAs). Over the past decade, rapidly emerging evidence has increasingly supported the view that lncRNAs serve key regulatory and functional roles in mammal cells. HIV-1 replication relies on various cell functions. To date, while the involvement of host protein factors and microRNAs (miRNAs) in the HIV-1 life cycle has been extensively studied, the relationship between lncRNAs and HIV-1 remains uncharacterized. Here, we have profiled 83 disease-related lncRNAs in HIV-1-infected T cells. We found NEAT1 to be one of several lncRNAs whose expression is changed by HIV-1 infection, and we have characterized its role in HIV-1 replication. We reported that the knockdown of NEAT1 enhances virus production through increased nucleus-to-cytoplasm export of Rev-dependent instability element (INS)-containing HIV-1 mRNAs.

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Chen, Chia-Yen; Liu, Xiang; Boris-Lawrie, Kathleen et al. (2013) Cellular RNA helicases and HIV-1: insights from genome-wide, proteomic, and molecular studies. Virus Res 171:357-65
Zhang, Quan; Jeang, Kuan-Teh (2013) Long non-coding RNAs (lncRNAs) and viral infections. Biomed Pharmacother 3:34-42
Zhang, Quan; Chen, Chia-Yen; Yedavalli, Venkat S R K et al. (2013) NEAT1 long noncoding RNA and paraspeckle bodies modulate HIV-1 posttranscriptional expression. MBio 4:e00596-12
Jerebtsova, Marina; Kumari, Namita; Xu, Min et al. (2012) HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells. Biology (Basel) 1:175-195
Houzet, Laurent; Klase, Zachary; Yeung, Man Lung et al. (2012) The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1. Nucleic Acids Res 40:11684-96
Tang, Sai-Wen; Ducroux, Aurelie; Jeang, Kuan-Teh et al. (2012) Impact of cellular autophagy on viruses: Insights from hepatitis B virus and human retroviruses. J Biomed Sci 19:92
Zheng, Yong-Hui; Jeang, Kuan-Teh; Tokunaga, Kenzo (2012) Host restriction factors in retroviral infection: promises in virus-host interaction. Retrovirology 9:112
Liu, Xiang; Houzet, Laurent; Jeang, Kuan-Teh (2012) Tombusvirus P19 RNA silencing suppressor (RSS) activity in mammalian cells correlates with charged amino acids that contribute to direct RNA-binding. Cell Biosci 2:41
Ammosova, Tatiana; Platonov, Maxim; Yedavalli, Venkat R K et al. (2012) Small molecules targeted to a non-catalytic ""RVxF"" binding site of protein phosphatase-1 inhibit HIV-1. PLoS One 7:e39481
Bachu, Mahesh; Yalla, Swarupa; Asokan, Mangaiarkarasi et al. (2012) Multiple NF-ýýB sites in HIV-1 subtype C long terminal repeat confer superior magnitude of transcription and thereby the enhanced viral predominance. J Biol Chem 287:44714-35

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