A widely-distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro
Kouki MatsudaSaiful IslamToru TakadaK. TsuchiyaBenjy Jek Yang TanShin‐ichiro HattoriHiroo KatsuyaKosaku KitagawaKwang Su KimMisaki MatsuoNicole S. DelinoHiroyuki GatanagaKazuhisa YoshimuraShuzo MatsushitaHiroaki MitsuyaShingo IwamiYorifumi SatouKenji Maeda
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ABSTRACT Persistence of HIV-1 latent reservoir cells during antiretroviral therapy is a major obstacle for curing HIV-1. Latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells; however, there are few useful models for evaluating LRA activity in vitro . Here, we established a long-term cell culture system harboring thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo . A combination of an LRA and antiretroviral therapy (ART) significantly reduced viral rebound upon treatment interruption. Experimental investigation and mathematical modeling demonstrated that addition of LRA to ART induced latency-reversing effect and contributed to the eradication of replication competent HIV-1. The widely distributed intact provirus elimination (WIPE) assay will be useful for optimizing therapeutics against HIV-1 latency and investigating mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.Keywords:
Provirus
Reversing
Virus latency
Reversion
Abstract Antiretroviral therapy has achieved great success in suppressing human immunodeficiency virus (HIV) replication and transforming HIV infection from a fatal disease to a manageable chronic disease. However, the latent HIV reservoir persists in the body of HIV-infected individuals and is prone to reactivation. Therefore, the development of new treatment methods aimed at a complete cure for HIV is needed. The leading strategy for HIV eradication is based on eliminating and preventing the reactivation of latent reservoirs through an approach known as “shock and kill.” This strategy involves the use of latency-reversing agents (LRAs) to activate the HIV provirus in latent viral reservoir cells. Many LRAs can be obtained from natural resources, including plants and marine organisms. In this review, we provide an overview of natural products used to eliminate HIV latency.
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Background Human Immunodeficiency Virus (HIV-1) persists in a latent state within resting CD4+ T cells of infected patients treated with highly active retroviral therapy (HAART). This latent reservoir should be eliminated for the clearance of infection. In these cells, silenced replication-competent viruses are integrated into the host genome and can be reactivated by T-cell stimuli. The establishment of this post-integration form of latency is a multifactorial process leading to transcriptional repression. The cellular and molecular mechanisms underlying HIV-1 promoter reactivation from latently infected cells are still poorly understood.
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In this thesis we focus on the ability of HIV-1 to establish a latent provirus in proliferating T cells and on how the silent provirus can be activated from latency. HIV-1 latency is a major barrier towards virus eradication from the infected individual. Knowledge on how the latent reservoir is established and understanding the molecular mechanisms that control latency may contribute to the development of therapeutics that aim at the prevention and/or eradication of proviral latency.
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The persistence of latent HIV-infected cellular reservoirs represents the major hurdle to virus eradication with highly active anti-retroviral therapy (HAART), since latently infected cells remain a permanent source of viral reactivation. HIV establishes a persistent infection in CD4+ T lymphocytes (and to a lesser extent in macrophages as well), creating a persistent reservoir consisting mainly of latently infected resting memory CD4+ T cell. Although pre- and post-integration latencies have been described in HIV-1, the reservoir that appears to be the major barrier to eradication is composed of latently infected cells carrying an integrated provirus that is transcriptionally silent.
It has been suggested that reactivation of the latent reservoirs could allow effective targeting and possible eradication of the virus. Immunoactivation therapy to reduce the latent pool of HIV by treatment with the anti-CD3 antibody OKT-3 alone or in combination with interleukin-2, substantially failed to significantly decrease the viral reservoir. Non-specific T-cell activation may induce high-level viral replication above a level that can be fully contained by ART, while increasing the susceptibility of uninfected cells.
Selective targeting of HIV provirus via agents that induce the expression of quiescent HIV, but have limited effects on the uninfected host cell is an alternate approach to attack latent HIV. Activation from latency to completion of the replication cycle should result in lytic cell death of CD4+ T cells. Multiple mechanisms that contribute to the maintenance of proviral latency could be targeted to activate the latent virus. As examples of potentially useful agents, IL–7 can reactivate HIV–1 in latently infected cells in vitro through the induction of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) signalling pathway. The use of different chemical compounds targeting the PKC signalling pathway (prostratin, bryostatin) has also been proposed as a means of reactivating viral reservoirs. Finally, HDAC blocking is an attractive potential means of inducing broad reactivation of HIV–1 reservoirs, and promising results have been achieved using the HDAC inhibitor vorinostat.
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Human immunodeficiency virus type 1 (HIV-1) infection is a chronic condition, where viral DNA integrates into the genome. Latently infected cells form a persistent, heterogeneous reservoir that at any time can reactivate the integrated HIV-1. Here we confirmed that latently infected cells from HIV-1 positive study participants exhibited active HIV-1 transcription but without production of mature spliced mRNAs. To elucidate the mechanisms behind this we employed primary HIV-1 latency models to study latency establishment and maintenance. We characterized proviral transcription and chromatin development in cultures of resting primary CD4+ T-cells for four months after ex vivo HIV-1 infection. As heterochromatin (marked with H3K9me3 or H3K27me3) gradually stabilized, the provirus became less accessible with reduced activation potential. In a subset of infected cells, active marks (e.g. H3K27ac) and elongating RNAPII remained detectable at the latent provirus, despite prolonged proviral silencing. In many aspects, latent HIV-1 resembled an active enhancer in a subset of resting cells. The enhancer chromatin actively promoted latency and the enhancer-specific CBP/P300-inhibitor GNE049 was identified as a new latency reversal agent. The division of the latent reservoir according to distinct chromatin compositions with different reactivation potential enforces the notion that even though a relatively large set of cells contains the HIV-1 provirus, only a discrete subset is readily able to reactivate the provirus and spread the infection.
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Human immunodeficiency virus type 1 (HIV-1) establishes a latent reservoir in resting memory CD4(+) T cells. This latent reservoir is a major barrier to the eradication of HIV-1 in infected individuals and is not affected by highly active antiretroviral therapy (HAART). Reactivation of latent HIV-1 is a possible strategy for elimination of this reservoir. The mechanisms with which latency is maintained are unclear. In the analysis of the regulation of HIV-1 gene expression, it is important to consider the nature of HIV-1 integration sites. In this study, we analyzed the integration and transcription of latent HIV-1 in a primary CD4(+) T cell model of latency. The majority of integration sites in latently infected cells were in introns of transcription units. Serial analysis of gene expression (SAGE) demonstrated that more than 90% of those host genes harboring a latent integrated provirus were transcriptionally active, mostly at high levels. For latently infected cells, we observed a modest preference for integration in the same transcriptional orientation as the host gene (63.8% versus 36.2%). In contrast, this orientation preference was not observed in acutely infected or persistently infected cells. These results suggest that transcriptional interference may be one of the important factors in the establishment and maintenance of HIV-1 latency. Our findings suggest that disrupting the negative control of HIV-1 transcription by upstream host promoters could facilitate the reactivation of latent HIV-1 in some resting CD4(+) T cells.
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While able to suppress HIV replication in HIV infected individuals, combination antiretroviral therapy (ART) fails to eliminate viral latent reservoir, which consists in integrated transcriptional silenced HIV provirus. So far, identification of latently-infected cells has relied on activating cells to induce expression of HIV proteins which can then be detected. Unfortunately, this activation significantly changed the cell phenotype. We developed a novel HIV reporter, named HIVGKO, that allows the purification of latently-infected cells in absence of reactivation. Indeed, latent cells can be identified by expression of the EF1a-driven mKO2 and lack of expression of the LTR-driven csGFP. This protocol can be used to study the effectiveness of LRAs (Latency Reversal Agents) in reactivating latent HIV in primary cells.
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The highly malignant line of morphologically transformed H-19 hamster tumor cells that harbor a single LTR, v-src, LTR provirus segregates morphologically flat revertants at the rate of 1.4 to 2.4 x 10(-3)/cell/cycle. Revertants behave like almost nonmalignant cells; they keep the provirus within an unaltered junction DNA fragment. However, the provirus is methylated, permanently transcriptionally silent, and not rescuable. Using the polymerase chain reaction, we have synthesized the whole proviral structure from two revertants and established that the left-hand long terminal repeats assuring transcription remained structurally intact. Moreover, the cloned proviral DNAs from three revertants were shown to produce tumors in chickens. The unusually high reversion rate together with the finding of structural integrity of proviral transcriptional signals in revertants indicate strongly that the reversion has been mediated by cell-regulatory mechanisms.
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