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Experimental Systems for Measuring HIV Latency and Reactivation

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Experimental Systems for Measuring HIV Latency and Reactivation viruses Review Experimental Systems for Measuring HIV Latency and Reactivation Koh Fujinaga 1,* and Daniele C. Cary 2 1 Division of Rheumatology, Department of Medicine, School of Medicine, University of California, San Francisco, CA 94143-0703, USA 2 Department of Medicine, Microbiology, and Immunology, School of Medicine, University of California, San Francisco, CA 94143-0703, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-415-502-1908 Received: 15 August 2020; Accepted: 5 November 2020; Published: 9 November 2020 Abstract: The final obstacle to achieving a cure to HIV/AIDS is the presence of latent HIV reservoirs scattered throughout the body. Although antiretroviral therapy maintains plasma viral loads below the levels of detection, upon cessation of therapy, the latent reservoir immediately produces infectious progeny viruses. This results in elevated plasma viremia, which leads to clinical progression to AIDS. Thus, if a HIV cure is ever to become a reality, it will be necessary to target and eliminate the latent reservoir. To this end, tremendous effort has been dedicated to locate the viral reservoir, understand the mechanisms contributing to latency, find optimal methods to reactivate HIV, and specifically kill latently infected cells. Although we have not yet identified a therapeutic approach to completely eliminate HIV from patients, these efforts have provided many technological breakthroughs in understanding the underlying mechanisms that regulate HIV latency and reactivation in vitro. In this review, we summarize and compare experimental systems which are frequently used to study HIV latency. While none of these models are a perfect proxy for the complex systems at work in HIV+ patients, each aim to replicate HIV latency in vitro. Keywords: HIV; latency; latency reversing agents; transcription; P-TEFb; silencing; transcription interference; NFkB 1. Introduction Antiretroviral therapy (ART) regimens keep the virus below the level of detection in HIV+ individuals. However, upon cessation of therapy, plasma viremia rapidly rebounds, leading to the progression to AIDS [1]. Although ART is successful at suppressing HIV, it is unable to completely eradicate the virus due to the presence of latent HIV reservoirs [2]. These reservoirs are long-lived viral sanctuaries scattered throughout the body. In HIV+ individuals, HIV-infected cells can be detected not only in the peripheral blood and lymph nodes, but also in the brain, lungs, kidneys, liver, adipose tissues, gastrointestinal tract, genitourinary systems, and bone marrow. Lymphoid tissues, such as spleen, thymus, and particularly gut-associated lymphoid tissues (GALT), are the most important viral reservoirs, which remain relatively dormant, but account for the immediate rebounds in viremia following cessation of ART [1,3]. One currently investigated approach to target the latent reservoir is therapeutic reactivation of HIV, also known as “shock and kill” [4,5]. A latency reversing agent (LRA) or combinations of LRAs would be used to reactivate and shock the virus out of hiding, leaving it vulnerable to immune killing machinery or cytolysis by the virus [4–7]. However, sufficient levels of HIV reactivation will be required for “shock and kill” therapies to significantly impact the reservoirs. Therefore, to develop the most effective reactivation strategies, it is essential to understand the precise cellular and molecular mechanisms by which HIV latency is established and controlled [8]. Viruses 2020, 12, 1279; doi:10.3390/v12111279 www.mdpi.com/journal/viruses Viruses 2020, 12, 1279 2 of 22 There are no animal models which fully recapitulate HIV pathogenesis as observed in HIV+ individuals [9]. Many studies on HIV latency and reactivation instead rely on tissue culture systems using immortalized human cell lines or primary cells derived from human blood or tissues [10,11]. These in vitro culture models utilize different combinations of growth factors, feeder cells, and cytokines to drive HIV-infected, activated T cells into a quiescent state, with the goal of modeling the conditions of latently infected T central or effector memory (Tcm or Tem) cells [12–14]. While all of these models produce resting T cells with transcriptionally silent HIV, it is unclear whether any of these in vitro latent HIV models phenocopy latent reservoirs in HIV+ individuals. In the following review, we present several stable cell line and primary cell models used to study HIV latency in order to provide a comprehensive comparison of the different available systems. 2. Mechanisms Regulating HIV Transcription and Latency HIV transcription is initiated at the transcription start site (TSS) located in the HIV long terminal repeat (LTR) region, which consists of a typical inducible promoter/enhancer sequences [15] (Figure1). Once integrated, the HIV LTR acts like a typical inducible cellular gene promoter, responding to various cellular signaling cues [15]. HIV transcription is regulated by host transcription factors (TFs) which control host gene expression. Following infection, activated cells can return to a resting state. These quiescent cells have vanishing low levels of important TFs. This maintains viral latency by keeping these cells in a transcriptionally silent state. Multiple cellular signaling pathways can control HIV transcription positively: positive transcription elongation factor b (P-TEFb), the nuclear factor kappa B (NFκB) pathway [16], the nuclear factor of activated T cells (NFAT) pathway [17], the mitogen-activated protein kinase (MAPK) pathway [18], the toll-like receptor (TLR) pathway [19], and the mammalian target of rapamycin (mTOR) pathway [20]); as well as negatively: via recruitment of the polycomb repressive complex (PRC) [21–23], the Cullin 3 E3 ligase pathway [24], and the zinc-finger protein 304 [25]. The proviral 50-LTR is an inducible promoter containing a TATA-box and upstream enhancer binding regions [26]. For example, a typical HIV LTR contains two (clade B) or three (clade C) NFκB sites and two SP1 sites [26]. Immediately after initiation of transcription and promoter clearance, the host RNA polymerase II (RNAPII) is stalled by the negative elongation factor (NELF) and DRB-sensitivity inducing factor (DSIF), resulting in accumulation of short premature RNAs with a length of less than 100 nucleotides that form a stem-loop structure called the transactivation response (TAR) element [27]. When there is sufficient HIV Tat, it binds to TAR and the cyclin T1 (CycT1) subunit of P-TEFb. The cyclin-dependent kinase 9 (CDK9) subunit of P-TEFb then hyperphosphorylates the C-terminal domain (CTD) of RNAPII at the second serine residues (S2) of the heptapeptide repeats (YSPTSPS), NELF and the SPT5 subunit of DSIF. NELF is then removed, and DSIF becomes a positive transcription factor, which accompanies RNAPII as it progresses through the gene body [28–31]. Recruitment of P-TEFb is a main switch between non-productive and productive transcription of HIV. Importantly, in quiescent CD4+ T cells, such as memory T cells [32,33] and hematopoietc stem cells [34–36], which represent the bulk of the latent reservoir, the expression of CycT1 is severely downregulated post-transcriptionally [37,38]. This downregulation of CycT1 causes a block of transcription elongation from the HIV LTR. In the late stage of HIV transcription, another cellular CDK/cyclin complex (CDK11/CycL) is recruited to RNAII elongation complex transcribing HIV [39,40]. CDK11 phosphorylates RNAPII’s S2 and recruits the transcription export (TREX/THO) complex, which promotes the cleavage and polyadenylation of HIV transcripts, stabilizing HIV mRNAs and enhancing their nuclear export and translation of viral proteins [39]. Interestingly, CDK11/CycL expression is also severely downregulated in quiescent CD4+ T cells [41]. Therefore, at least two major cellular CDK/cyclin complexes play key roles in HIV latency [42] (Figure1). Recent studies from Yukl and colleagues demonstrated that HIV transcription is blocked at multiple steps including initiation, elongation, splicing, termination, and polyadenylation [43]. Viruses 2020, 12, 1279 3 of 22 Viruses 2020, 12, x FOR PEER REVIEW 3 of 23 Figure 1. Two major cellular cyclin/Cdk complexes play key roles in HIV transcription. P-TEFb Figure 1. Two major cellular cyclin/Cdk complexes play key roles in HIV transcription. P-TEFb (CycT1:CDK9, green) is recruited to the transcription machinery on HIV LTR by various factors. Epigenetic(CycT1:CDK9, factor green) Brd4, is DNA-boundrecruited to the transactivator transcription NF machineryκB, and viral on HIV Tat (associatedLTR by various with cellularfactors. κ superEpigenetic elongation factor Brd4, complex) DNA-bound bind to CycT1 transactivator and recruit NF P-TEFbB, and toviral HIV Tat provirus (associated in an with active cellular chromatin super environment.elongation complex) P-TEFb thenbind phophorylatesto CycT1 and RNAPII recruit andP-TEFb negative to HIV transcription provirus elongationin an active factors chromatin NELF andenvironment. DSIF, which P-TEFb augments then transcriptional phophorylates elongation. RNAPII CycL:Cdk11and negative complex transcription (yellow), elongation in turn, associated factors withNELF transcription and DSIF, which/export augments and THO tr complexanscriptional (TREX elongation./THOC) is CycL:C recruiteddk11 to complex RNAPII transcribing(yellow), in turn, HIV genes.associated Cdk11 with phosphorylates transcription/export the CTD and of THO RNAPII complex and promotes(TREX/THOC)
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