Sekar Natesampillai

A Novel Functional Interaction between the Sp1-like Protein KLF13 and SREBP-Sp1 Activation Complex Underlies Regulation of Low Density Lipoprotein Receptor Promoter Function

Cholesterol homeostasis is regulated by a family of transcription factors designated sterol regulatory element-binding proteins (SREBPs). Precise control of SREBP-targeted genes requires additional interactions with co-regulatory transcription factors. In the case of the low density lipoprotein receptor (LDLR), SREBP cooperates with the specificity protein Sp1 to activate the promoter. In this report, we describe a novel pathway in LDLR transcriptional regulation distinct from the SREBP-Sp1 activation complex involving the Sp1-like protein Krueppel-like factor 13 (KLF13). Using a combination of RNA interference, electrophoretic mobility shift, chromatin immunoprecipitation, and reporter assays, deletion, and site-directed mutagenesis, we demonstrated that KLF13 mediates repression in a DNA context-selective manner. KLF13 repression of LDLR promoter activity appears to be needed to keep the receptor silent, a state that can be antagonized by Sp1, SREBP, and inhibitors of histone deacetylase activity. Chromatin immunoprecipitation assay confirmed that KLF13 binds proximal LDLR DNA sequences in vivo and that exogenous oxysterol up-regulates such binding. Together these studies identify a novel regulatory pathway in which gene repression by KLF13 must be overcome by the Sp1-SREBP complex to activate the LDLR promoter. Therefore, these data should replace a pre-existent and more simple paradigm that takes into consideration only the induction of the activator proteins Sp1-SREBP as necessary for LDLR promoter drive without including default repression, such as that by KLF13, of the LDLR gene.

The HIV-1-Specific Protein Casp8p41 Induces Death of Infected Cells through Bax/Bak

ABSTRACT Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.

Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells

BackgroundHIV eradication strategies are now being evaluated in vitro and in vivo. A cornerstone of such approaches is maximal suppression of viral replication with combination antiretroviral therapy (ART). Since many antiretroviral agents have off target effects, and different classes target different components of the viral life cycle, we questioned whether different classes of ART might differentially affect the survival and persistence of productively HIV-infected CD4 T cells.MethodsIn vitro infections of primary CD4 T cells using clinical isolates of HIV-1 that were either protease inhibitor susceptible (HIV PI-S), or resistant (HIV PI-R) were treated with nothing, lopinavir, efavirenz or raltegravir. Cell viability, apoptosis, and the proportion of surviving cells that were P24 positive was assessed by flow cytometry.ResultsIn HIV PI-S infected primary cultures, all three antiretroviral agents decreased viral replication, and reduced the total number of cells that were undergoing apoptosis (P < 0.01) similarly. Similarly, in the HIV PI-R infected cultures, both efavirenz and raltegravir reduced viral replication and reduced apoptosis compared to untreated control (P < 0.01), while lopinavir did not, suggesting that HIV replication drives T cell apoptosis, which was confirmed by association by linear regression (P < 0.0001) . However since HIV protease has been suggested to directly induce apoptosis of infected CD4 T cells, and HIV PI are intrinsically antiapoptotic, we evaluated apoptosis in productively infected (HIV P24+) cells. More HIV p24 positive cells were apoptotic in the Efavirenz or raltegravir treated cultures than the lopinavir treated cultures (P = 0.0008 for HIV PI-R and P = 0.06 for the HIV PI-S), indicating that drug class impacts survival of productively infected CD4 T cells.ConclusionsInhibiting HIV replication with a PI, NNRTI or INSTI reduces total HIV-induced T cell apoptosis. However, blocking HIV replication with PI but not with NNRTI or INSTI promotes survival of productively HIV-infected cells. Thus, selection of antiretroviral agents may impact the success of HIV eradication strategies.

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation

HIV protease converts procaspase 8 into Casp8p41, which binds and activates Bak to induce cell death in infected CD4 T cells.

Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells.

In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study

Background Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. Methods and findings We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures—including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue—the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. Conclusions allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.

Isolation of a TRAIL Antagonist from the Serum of HIV-infected Patients

Background: The TRAIL:TRAIL receptor system has been implicated in the pathogenesis of a variety of malignant and infectious disorders, including HIV infection. Results: We show that HIV causes production of a novel TRAIL splice variant, that we call TRAIL-short, which binds TRAIL R2, antagonizes TRAIL signaling, and is present in HIV patient samples. Conclusion: Introduction of TRAIL-short causes resistance to TRAIL, whereas knockdown restores sensitivity. Significance: The identification of TRAIL-short impacts our understanding of TRAIL sensitivity and has implications for the pathogenesis of both infectious and malignant pathogenesis. Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.

Maintenance of the HIV reservoir is antagonized by selective BCL2 inhibition

ABSTRACT Decay of the HIV reservoir is slowed over time in part by expansion of the pool of HIV-infected cells. This expansion reflects homeostatic proliferation of infected cells by interleukin-7 (IL-7) or antigenic stimulation, as well as new rounds of infection of susceptible target cells. As novel therapies are being developed to accelerate the decay of the latent HIV reservoir, it will be important to identify interventions that prevent expansion and/or repopulation of the latent HIV reservoir. Our previous studies showed that HIV protease cleaves the host protein procaspase 8 to generate Casp8p41, which can bind and activate Bak to induce apoptosis of infected cells. In circumstances where expression of the anti-apoptotic protein BCL2 is high, Casp8p41 instead binds BCL2, and cell death does not occur. This effect can be overcome by treating cells with the clinically approved BCL2 antagonist venetoclax, which prevents Casp8p41 from binding BCL2, thereby allowing Casp8p41 to bind Bak and kill the infected cell. Here we assess whether the events that maintain the HIV reservoir are also antagonized by venetoclax. Using the J-Lat 10.6 model of persistent infection, we demonstrate that proliferation and HIV expression are countered by the use of venetoclax, which causes preferential killing of the HIV-expressing cells. Similarly, during new rounds of infection of primary CD4 T cells, venetoclax causes selective killing of HIV-infected cells, resulting in decreased numbers of HIV DNA-containing cells. IMPORTANCE Cure of HIV infection requires an intervention that reduces the HIV reservoir size. A variety of approaches are being tested for their ability to impact HIV reservoir size. Even if successful, however, these approaches will need to be combined with additional complementary approaches that prevent replenishment or repopulation of the HIV reservoir. Our previous studies have shown that the FDA-approved BCL2 antagonist venetoclax has a beneficial effect on the HIV reservoir size following HIV reactivation. Here we demonstrate that venetoclax also has a beneficial effect on HIV reservoir size in a model of homeostatic proliferation of HIV as well as in acute spreading infection of HIV in primary CD4 T cells. These results suggest that venetoclax, either alone or in combination with other approaches to reducing HIV reservoir size, is a compound worthy of further study for its effects on HIV reservoir size.

TRAIL Dependent Fratricidal Killing of gp120 Primed Hepatocytes by HCV Core Expressing Hepatocytes

The mechanism by which HIV and HCV cooperatively accelerate hepatocyte damage is not clearly understood; however, each virus affects the TRAIL: TRAIL-receptor system. We, therefore, questioned whether the independent effects of HCV and HIV combine to synergistically result in TRAIL dependent hepatocyte killing. We describe that Huh7 hepatocytes treated with HIV gp120 results in both increase TRAIL-R2 expression and an acquired sensitivity to TRAIL mediated killing. Moreover HCV infection and HCV core expression alone in Huh7 cells upregulates TRAIL. Co-incubation of HIV gp120 primed hepatocytes with HCV core expressing hepatocytes results in the selective death of the HIV gp120 primed hepatocytes that is selectively blocked by TRAIL-R2-Fc fusion protein. Liver biopsies from HIV mono-infected patients have increased TRAIL-R2; biopsies from HCV infected patients have increased TRAIL, while co-infected liver biopsies have increased PARP cleavage within hepatocytes indicating enhanced apoptosis. These findings suggest a pathogenic model to understand why HIV/HCV co-infection accelerates liver injury.

Actions of Insulin and Insulin-Like Growth Factor-1 on Sterol-Metabolizing Gene Expression in Ovarian Cells

The mammalian female reproductive cycle is an intricately regulated process. In normal physiology, insulin-like peptides and gonadotropic hormones such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH) initiate signaling cascades that are critical for follicular growth, ovulation, luteinization, steroidogenesis, fertilization, and successful implantation. The pituitary gonadotropins, FSH and LH, play primary roles in directing steroid-hormone biosynthesis and cognate gene expression. In addition, insulin, insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), members of the transforming growth factor-beta (TGF-β) family (e.g., inhibin, activin, follistatin), bone morphogenetic protein (BMG), and growth differentiation factor (GDF) contribute to cytodifferen-tiative control. This chapter highlights the physiological importance of insulin and IGF-1 in steroidogenic gene regulation where relevant transgenic (murine) models are illustrated.