Himmelfarb Health Sciences Library, The George Washington University Health Sciences Research Commons Medicine Faculty Publications Medicine 5-1-2018 Tat controls transcriptional persistence of unintegrated HIV genome in primary human macrophages. Beatrix Meltzer Deemah Dabbagh Jia Guo Fatah Kashanchi Mudit Tyagi George Washington University See next page for additional authors Follow this and additional works at: https://hsrc.himmelfarb.gwu.edu/smhs_medicine_facpubs Part of the Virology Commons, and the Virus Diseases Commons APA Citation Meltzer, B., Dabbagh, D., Guo, J., Kashanchi, F., Tyagi, M., & Wu, Y. (2018). Tat controls transcriptional persistence of unintegrated HIV genome in primary human macrophages.. Virology, 518 (). http://dx.doi.org/10.1016/j.virol.2018.03.006 This Journal Article is brought to you for free and open access by the Medicine at Health Sciences Research Commons. It has been accepted for inclusion in Medicine Faculty Publications by an authorized administrator of Health Sciences Research Commons. For more information, please contact [email protected]. Authors Beatrix Meltzer, Deemah Dabbagh, Jia Guo, Fatah Kashanchi, Mudit Tyagi, and Yuntao Wu This journal article is available at Health Sciences Research Commons: https://hsrc.himmelfarb.gwu.edu/smhs_medicine_facpubs/ 1210 Virology 518 (2018) 241–252 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/virology Tat controls transcriptional persistence of unintegrated HIV genome in T primary human macrophages Beatrix Meltzera,1,2, Deemah Dabbagha,2, Jia Guoa,3, Fatah Kashanchib, Mudit Tyagia,4, ⁎ Yuntao Wua, a National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, USA b Laboratory of Molecular Virology, George Mason University, Manassas, USA ARTICLE INFO ABSTRACT Keywords: In HIV infected macrophages, a large population of viral genomes persists as the unintegrated form (uDNA) that HIV is transcriptionally active. However, how this transcriptional activity is controlled remains unclear. In this re- Unintegrated HIV DNA port, we investigated whether Tat, the viral transactivator of transcription, is involved in uDNA transcription. Macrophage We demonstrate that de novo Tat activity is generated from uDNA, and this uDNA-derived Tat (uTat) transac- Transcription tivates the uDNA LTR. In addition, uTat is required for the transcriptional persistence of uDNA that is assembled Tat into repressive episomal minichromatin. In the absence of uTat, uDNA minichromatin is gradually silenced, but Persistence Reservoir remains highly inducible by HDAC inhibitors (HDACi). Therefore, functionally, uTat antagonizes uDNA mini- HDACi chromatin repression to maintain persistent viral transcription in macrophages. uTat-mediated viral persistence may establish a viral reservoir in macrophages where uDNA were found to persist. 1. Introduction activation (Fenard et al., 2005; Schrager and Marsh, 1999; Wu and Marsh, 2001). This early Nef from uDNA can also effectively down- The natural process of HIV infection leads to the accumulation of a modulate surface receptors such as CD4, CXCR4/CCR5, and MHC class I large amount of unintegrated viral DNA (uDNA) in CD4 T cells, mac- (Gillim-Ross et al., 2005b; Sloan et al., 2011a, 2011b). In addition, rophages, lymphoid tissues, and the brain (Chun et al., 1997; Pang uDNA can stimulate the production of numerous inflammatory che- et al., 1990; Pauza et al., 1990; Shaw et al., 1984; Teo et al., 1997). In mokines such as CXCL8 (IL-8), CXCL9, and CXCL10 (IP-10) in macro- cell culture conditions, uDNA has been found to possess transcriptional phages (Kelly et al., 2008). The persistence of uDNA in immune cells, activity, which can generate all three classes of viral transcripts: the such as blood resting CD4 T cells and macrophages, has also been multiply spliced, the singly spliced, and the non-spliced (Kelly et al., suggested to serve as alternative form of viral reservoirs, which can be 2008; Wu and Marsh, 2001, 2003). Nevertheless, only viral early pro- readily reactivated by T cell activation, cytokines, gut-associated short teins such as Nef are selectively translated in primary T cells and chain fatty acids, or other latency reversing agents for gene expression, macrophages in the absence of cellular stimulation (Kelly et al., 2008; and even low level viral replication (Chan et al., 2016; Gelderblom Wu and Marsh, 2001, 2003). This selective transcription has been et al., 2008; Kantor et al., 2009; Petitjean et al., 2007; Trinite et al., suggested to be a normal early process of HIV infection of human T cells 2013; Wu, 2008). In addition, genetic complementation between un- and macrophages (Kelly et al., 2008; Wu, 2004; Wu and Marsh, 2001, integrated and integrated viral genomes can occur, and this process has 2003). In particular, in cultured macrophages, uDNA can persist for at been suggested to be essential to prevent possible losses of viral genetic least 30 days and remain transcriptionally active at a low level (Gillim- diversity (Gelderblom et al., 2008; Wu, 2008). Thus, the two tran- Ross et al., 2005a; Kelly et al., 2008). scriptional processes, occurring before and after HIV integration, are Functionally, the production of Nef from uDNA has been suggested intimately linked to facilitate viral infection, latency, and persistence to facilitate HIV-1 infection by lowering the threshold of T cell (Wodarz et al., 2014; Wu, 2008). ⁎ Corresponding author. E-mail address: [email protected] (Y. Wu). 1 Present address: Children’s National Health System, Department of Lab Medicine, Washington DC, USA. 2 Both authors contributed equally to this work. 3 Present address: Merck Sharp & Dohme Corp, Boston, USA. 4 Present address: The George Washington University, Washington DC, USA. https://doi.org/10.1016/j.virol.2018.03.006 Received 2 December 2017; Received in revised form 6 March 2018; Accepted 7 March 2018 Available online 15 March 2018 0042-6822/ © 2018 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). B. Meltzer et al. Virology 518 (2018) 241–252 Recent studies have also suggested that preintegration transcription and Rev-CEM-GFP-Luc (Wu et al., 2007a, 2007b) (Virongy, Manassas, occurs at a large scale, and the number of transcribing uDNA approx- VA). NL4-3(KFS) and NL(KFS)(D116N) were produced by cotransfec- imates to those of proviral genomes (Iyer et al., 2009). However, each tion of HEK293T cells with pHCMV-G and either pNL4-3(KFS) or pNL4- uDNA template transcribes at a homogenously low level (Iyer et al., 3(KFS)(D116N) in a 1:1 ratio. vLTR-Tat-IRES-GFP and vLTR-Tat-IRES- 2009). This raises the question of whether this low level transcription is GFP(D116N) were produced by cotransfection of HEK293T cells with regulated by Tat, the viral transactivator of transcription. Tat stimulates pLTR-Tat-IRES-GFP, pHCMV-G, and either pCMVΔR8.2 or viral gene expression by binding to the transactivation response (TAR) pCMVΔR8.2(D116N) in a ratio of 4:1:3 respectively. vLTR-GFP and element on RNA and recruits factors such as cyclin T1 and CDK9 (P- vLTR-GFP(D116N) were produced by cotransfection of HEK293T cells TEFb) for transcriptional initiation and polymerase processivity with pLTR-GFP, pHCMV-G, and either pCMVΔR8.2 or (Herrmann and Rice, 1993, 1995; Mancebo et al., 1997; Wei et al., pCMVΔR8.2(D116N) in a ratio of 4:1:3, respectively. vNL-Luc and virus 1998; Zhu et al., 1997). Tat can also directly bind to components of the vNL-Luc(D116N) were constructed by co-transfection of HEK293T cells ATP-dependent chromatin remodeling complex SWI-SNF (Agbottah with pLTR-Luc, pHCMV-G, and either pCMVΔR8.2 or et al., 2006; Mahmoudi et al., 2006; Treand et al., 2006) and recruits pCMVΔR8.2(D116N) in a 4:1:3 ratio, respectively. vLTR-Tat-IRES-Luc the complex to the HIV promoter (Agbottah et al., 2006; Mahmoudi and vLTR-Tat-IRES-Luc(D116N) were generated from co-transfection of et al., 2006; Treand et al., 2006). In addition, Tat can bind to HAT HEK293T cells with pLTR-Tat-IRES-Luc, pHCMV-G, and either (histone acetyltransferase) such as p300/CBP, pCAF, and hGCN5 pCMVΔR8.2 or pCMVΔR8.2(D116N) in a 4:1:3 ratio, respectively. Viral (Benkirane et al., 1998; Col et al., 2001; Hottiger and Nabel, 1998; supernatant was harvested at 48 h postcotransfection, filtered through a Kamine et al., 1996; Marzio et al., 1998), which may promote acet- 0.45-μm filter, and stored at −80 oC. Levels of p24 in viral supernatant ylation of nucleosomes and Tat itself to enhance Tat-mediated trans- were measured using a Perkin Elmer Alliance p24 antigen enzyme- activation (Gatignol, 2007; Kiernan et al., 1999). Nevertheless, given linked immunosorbent assay kit (Perkin Elmer, Waltham, MA) or an in- the low transcriptional activity of uDNA, the role of Tat in activating house p24 antigen ELISA kit. Viral infection of T cells and macrophages uDNA transcription remained uncertain. In this article, we examined were carried out as previously described (Kelly et al., 2008; Yoder et al., the role of Tat in regulating uDNA transcription and viral persistence in 2008). HIV infection was also carried through spinoculation (Guo et al., primary macrophages. 2011), in which viral particles were added to the cells and subjected to centrifugation for 2 h at 600 ×g (Guo et al., 2011). After centrifugation, 2. Materials and methods the medium was replaced with 1 ml fresh RPMI 1640 medium supple- mented with 10% heat-inactivated FBS and incubated at 37 oC for an- 2.1. Approvals from IRB and IACUC other 48 h. Peripheral blood was drawn from HIV-negative donors. All proto- cols involving human subjects were reviewed and approved by the 2.4. Cells and cell culture George Mason University (GMU) IRB. Informed written consents from the human subjects were obtained in this study. Rev-dependent cell line Rev-CEM-GFP (Wu et al., 2007a, 2007b), CEM-SS cells (Virongy, Manassas, VA), and U937 cells (from NIH AIDS 2.2.