Amy S. Espeseth

Genome-scale RNAi screen for host factors required for HIV replication.

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

Host Cell Factors in HIV Replication: Meta-Analysis of Genome-Wide Studies

We have analyzed host cell genes linked to HIV replication that were identified in nine genome-wide studies, including three independent siRNA screens. Overlaps among the siRNA screens were very modest (<7% for any pairwise combination), and similarly, only modest overlaps were seen in pairwise comparisons with other types of genome-wide studies. Combining all genes from the genome-wide studies together with genes reported in the literature to affect HIV yields 2,410 protein-coding genes, or fully 9.5% of all human genes (though of course some of these are false positive calls). Here we report an “encyclopedia” of all overlaps between studies (available at http://www.hostpathogen.org), which yielded a more extensively corroborated set of host factors assisting HIV replication. We used these genes to calculate refined networks that specify cellular subsystems recruited by HIV to assist in replication, and present additional analysis specifying host cell genes that are attractive as potential therapeutic targets.

Diketo acid inhibitor mechanism and HIV-1 integrase: Implications for metal binding in the active site of phosphotransferase enzymes

The process of integrating the reverse-transcribed HIV-1 DNA into the host chromosomal DNA is catalyzed by the virally encoded enzyme integrase (IN). Integration requires two metal-dependent reactions, 3′ end processing and strand transfer. Compounds that contain a diketo acid moiety have been shown to selectively inhibit the strand transfer reaction of IN in vitro and in infected cells and are effective as inhibitors of HIV-1 replication. To characterize the molecular basis of inhibition, we used functional assays and binding assays to evaluate a series of structurally related analogs. These studies focused on investigating the role of the conserved carboxylate and metal binding. We demonstrate that an acidic moiety such as a carboxylate or isosteric heterocycle is not required for binding to the enzyme complex but is essential for inhibition and confers distinct metal-dependent properties on the inhibitor. Binding requires divalent metal and resistance is metal dependent with active site mutants displaying resistance only when the enzymes are evaluated in the context of Mg2+. The mechanism of action of these inhibitors is therefore likely a consequence of the interaction between the acid moiety and metal ion(s) in the IN active site, resulting in a functional sequestration of the critical metal cofactor(s). These studies thus have implications for modeling active site inhibitors of IN, designing and evaluating analogs with improved efficacy, and identifying inhibitors of other metal-dependent phosphotransferases.

Expression of Latent HIV Induced by the Potent HDAC Inhibitor Suberoylanilide Hydroxamic Acid

Histone deacetylases (HDACs) act on histones within the nucleosome-bound promoter of human immunodeficiency virus type 1 (HIV-1) to maintain proviral latency. HDAC inhibition leads to promoter expression and the escape of HIV from latency. We evaluated the ability of the potent inhibitor recently licensed for use in oncology, suberoylanilide hydroxamic acid (SAHA; Vorinostat), selective for Class I HDACs, to induce HIV promoter expression in cell lines and virus production from the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients. In J89, a Jurkat T cell line infected with a single HIV genome encoding the enhanced green fluorescence protein (EGFP) within the HIV genome, SAHA induced changes at nucleosome 1 of the HIV promoter in chromatin immunoprecipitation (ChIP) assays in concert with EGFP expression. In the resting CD4(+) T cells of antiretroviral-treated, aviremic HIV-infected patients clinically achievable exposures to SAHA induced virus outgrowth ex vivo. These results suggest that potent, selective HDAC inhibitors may allow improved targeting of persistent proviral HIV infection, and define parameters for in vivo studies using SAHA.

c-Myc and Sp1 Contribute to Proviral Latency by Recruiting Histone Deacetylase 1 to the Human Immunodeficiency Virus Type 1 Promoter

ABSTRACT Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4+ T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

A Limited Group of Class I Histone Deacetylases Acts To Repress Human Immunodeficiency Virus Type 1 Expression

ABSTRACT Silencing of the integrated human immunodeficiency virus type 1 (HIV-1) genome in resting CD4+ T cells is a significant contributor to the persistence of infection, allowing the virus to evade both immune detection and pharmaceutical attack. Nonselective histone deacetylase (HDAC) inhibitors are capable of inducing expression of quiescent HIV-1 in latently infected cells. However, potent global HDAC inhibition can induce host toxicity. To determine the specific HDACs that regulate HIV-1 transcription, we evaluated HDAC1 to HDAC11 RNA expression and protein expression and compartmentalization in the resting CD4+ T cells of HIV-1-positive, aviremic patients. HDAC1, -3, and -7 had the highest mRNA expression levels in these cells. Although all HDACs were detected in resting CD4+ T cells by Western blot analysis, HDAC5, -8, and -11 were primarily sequestered in the cytoplasm. Using chromatin immunoprecipitation assays, we detected HDAC1, -2, and -3 at the HIV-1 promoter in Jurkat J89GFP cells. Targeted inhibition of HDACs by small interfering RNA demonstrated that HDAC2 and HDAC3 contribute to repression of HIV-1 long terminal repeat expression in the HeLa P4/R5 cell line model of latency. Together, these results suggest that HDAC inhibitors specific for a limited number of class I HDACs may offer a targeted approach to the disruption of persistent HIV-1 infection.

Expression of latent human immunodeficiency type 1 is induced by novel and selective histone deacetylase inhibitors

Objectives:A family of histone deacetylases (HDACs) mediates chromatin remodeling, and repression of gene expression. Deacetylation of histones within the HIV-1 long terminal repeat (LTR) by HDACs plays a key role in the maintenance of latency, whereas acetylation of histones about the LTR is linked to proviral expression and escape of HIV from latency. Global HDAC inhibition may adversely affect host gene expression, leading to cellular toxicities. Potent inhibitors selective for HDACs that maintain LTR repression could be ideal antilatency therapeutics. Methods:We investigated the ability of selective HDAC inhibitors to de-repress the HIV-1 LTR in both a cell line model of latency and in resting CD4+ T cells isolated from patients who were aviremic on antiretroviral therapy (ART). Results:We found that inhibition of class I HDACs increased acetylation of histones at the LTR, but that LTR chromatin was unaffected by class II HDAC inhibitors. In a latently infected cell line, inhibitors selective for class I HDACs were more efficient activators of the LTR than inhibitors that target class II HDACs. Class I HDAC inhibitors were strikingly efficient inducers of virus outgrowth from resting CD4+ T cells of aviremic patients, whereas HIV was rarely recovered from patients cells exposed to class II HDAC inhibitors. Conclusions:Further development of selective HDAC inhibitors as part of a clinical strategy to target persistent HIV infection is warranted.

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

Robust statistical methods for hit selection in RNA interference high-throughput screening experiments

RNA interference (RNAi) high-throughput screening (HTS) experiments carried out using large (>5000 short interfering [si]RNA) libraries generate a huge amount of data. In order to use these data to identify the most effective siRNAs tested, it is critical to adopt and develop appropriate statistical methods. To address the questions in hit selection of RNAi HTS, we proposed a quartile-based method which is robust to outliers, true hits and nonsymmetrical data. We compared it with the more traditional tests, mean +/- k standard deviation (SD) and median +/- 3 median of absolute deviation (MAD). The results suggested that the quartile-based method selected more hits than mean +/- k SD under the same preset error rate. The number of hits selected by median +/- k MAD was close to that by the quartile-based method. Further analysis suggested that the quartile-based method had the greatest power in detecting true hits, especially weak or moderate true hits. Our investigation also suggested that platewise analysis (determining effective siRNAs on a plate-by-plate basis) can adjust for systematic errors in different plates, while an experimentwise analysis, in which effective siRNAs are identified in an analysis of the entire experiment, cannot. However, experimentwise analysis may detect a cluster of true positive hits placed together in one or several plates, while platewise analysis may not. To display hit selection results, we designed a specific figure called a plate-well series plot. We thus suggest the following strategy for hit selection in RNAi HTS experiments. First, choose the quartile-based method, or median +/- k MAD, for identifying effective siRNAs. Second, perform the chosen method experimentwise on transformed/normalized data, such as percentage inhibition, to check the possibility of hit clusters. If a cluster of selected hits are observed, repeat the analysis based on untransformed data to determine whether the cluster is due to an artifact in the data. If no clusters of hits are observed, select hits by performing platewise analysis on transformed data. Third, adopt the plate-well series plot to visualize both the data and the hit selection results, as well as to check for artifacts.

LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease

Rare familial forms of Alzheimers disease (AD) are thought to be caused by elevated proteolytic production of the Aβ42 peptide from the β-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Aβ42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Aβ42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Aβ40, Aβ42, and sAPPβ, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Aβ secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Aβ42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.