Title: The role of LEDGF/p75 in HIV replication Analysis of knockdown and knockout cell lines and translational implications
Other Titles: De rol van LEDGF/p75 in HIV replicatie Analyse van knockdown en knockout cellijnen and translationele implicaties
Authors: Schrijvers, Rik
Issue Date: 7-Dec-2012
Abstract: HIV, the causative agent of AIDS affects more than 34 million people worldwide. Controlling the HIV pandemic – and ultimately curing HIV – remains one of the major challenges in medicine. Although antiretroviral drugs have markedly changed the outcome of HIV infected patients, converting a deadly disease into a chronic infection, they are not without important problems and drawbacks. Even with the most effective antiretroviral drug combinations, treatment is presumed to be required life-long, can give rise to various side effects and is prone to rapid resistance development in case of sub-optimal adherence. In addition, a vaccine likely will not be available in the coming years despite decades of research. In this context, efforts on gaining further insights in retroviral biology and valorization of possible new targets to tackle HIV remain indispensable. This thesis dissertation focused on the role of the host cell protein Lens epithelium-derived growth factor (LEDGF/p75) in the HIV replication cycle, and related these findings with the recently developed LEDGINs, a new class of antiretroviral drugs targeting the viral IN, and with genetic LEDGF/p75 variants observed in patients.Integration of viralDNA into the host cell genome is a critical step during HIV replication. A stably inserted provirus is essential for productive infection and archives the genetic information of HIV in the host cell. The presence ofa permanent viral reservoir that evades the immune system and enables HIV to rebound once antiretroviral therapy is ceased is one of the major hurdles remaining to surmount the HIV epidemic. Lentiviral integration is catalysed by the viral enzyme integrase in close association with the cellular cofactor LEDGF/p75. RationaleOur group and others have elaborated extensively on the role of LEDGF/p75 in HIV replication.It is key to put the present study in its historical context to apprehend its objectives and methodological rationale. In 2003, LEDGF/p75 was identified as a binding partner of HIV integrase in our group and 3 yearslater, depletion of LEDGF/p75 in cell culture was shown to hamper HIV replication. Initially, this could not be confirmed by other groups questioning the role of LEDGF/p75 in HIV replication. From hindsight, this controversy could be attributed to the limitations of the RNAi technology,since minute amounts of LEDGF/p75 were shown to be sufficient to support HIV replication. However, even upon potent LEDGF/p75 knockdown, residual replication was observed, leaving open whether this co-factor was essential. In the meantime, molecular studies elucidated the interaction interface of LEDGF/p75 and integrase and indicated that the pocket formed by an integrase dimer that binds LEDGF/p75 could be targeted by small molecules. Enthusiasm to develop these small molecules was thwarted in 2007, when a study in mouse embryonic fibroblasts was published, demonstrating that residual HIV-derived vector transduction was still possible despite Psip1 knockout, suggesting that LEDGF/p75 was a non-essential co-factor for HIV replication. However, these studies were performed in mouseembryonic fibroblast knocked out for both LEDGF/p52 and LEDGF/p75 – mouse nor fibroblasts are classical targets of HIV – and thus researchers relied on single round HIV-derived vectors and not replicating virus for these experiments. At that moment, we decided in our group to generate ahuman somatic LEDGF/p75 knockout cell line, enabling the study of multiple round HIV replication in the complete absence of LEDGF/p75 in a human context.ObjectivesThe first goal of this study was to generateand validate a human LEDGF/p75 knockout cell line. Next, this cell model would enable us to answer our main scientific questions: 1) is LEDGF/p75 essential for HIV replication; 2) if not, how can HIV replicate in the absence of LEDGF/p75; 3) can HIV adapt to the absence of LEDGF/p75; 4)how do these results translate to small molecules targeting the IN-LEDGF/p75 interaction interface. In addition, this model could provide additional clues for the HIV integration site selection process and help in the elucidation of the cellular role(s) of LEDGF/p75.ResultsIn the first chapter of this thesis we describe the generation and validationof a LEDGF/p75 knockout cell line. By homologous recombination the exons encoding the LEDGF/p75 IBD were knocked out in a human Nalm-6 cell line, leaving the LEDGF/p52 splice variant unaltered. In the absence of LEDGF/p75, replication of laboratory HIV-1 strains was shown to be severelydelayed, yet virus still replicated, while replication of clinical HIV-1 isolates was undetectable. More detailed analysis showed that the residual replication was mediated by Hepatoma-derived growth factor related protein 2 (HRP-2), the only cellular protein besides LEDGF/p75 that contains an IBD. The IN-LEDGF/p75 inhibitors (LEDGINs) remained active even in the absence of LEDGF/p75 and blocked the interaction with the IBD of both LEDGF/p75 and HRP-2. In the second chapter we elaborate further on the role of LEDGF/p75 in the integration site selection process. This work and previous work demonstrated that LEDGF/p75 depletion significantly altered the lentiviral integration site distribution, shiftingintegration out of transcription units. However, the integration pattern in the absence of LEDGF/p75 remained distinct from a computationally generated matched random control set, suggesting the presence of an alternative tethering factor. We evaluated the role of HRP-2 in the integration site selection process using LEDGF/p75 and/or HRP-2 depleted cells. Our results indicated that additional HRP-2 depletion in LEDGF/p75-depleted cells further reduced integration frequency in transcription units and shifted the integration site distribution pattern more towards random.Our results demonstrated that the residual bias in integration targeting observed in the absence of LEDGF/p75 can, at least in part, be ascribed to HRP-2. Depletion of HRP-2 alone however did not affect the HIV-1 integration pattern, underscoring the dominance of LEDGF/p75 as HIV-1 INco-factor.In the third chapter we evaluate whether LEDGF/p75 plays a role during the late steps of the viral replication cycle, this is duringpost-integration steps. The rationale for this study stems from the observation that LEDGINs (compounds that bind the viral IN and inhibit the interaction with the cellular LEDGF/p75) inhibit both pre- and post-integration steps. Our model is the first to provide the means to study thisquestion in detail. We unveiled an unknown role of LEDGF/p75 in HIV replication by demonstrating that virions produced in LEDGF/p75 KO cells displayed a reduced infectivity in a subsequent round of infection. The effect was pinpointed to post-entry steps, presumably steps early after infection.In the fourth chapter we use our cell model to evaluate genetic LEDGF/p75 variants, associated with altered disease progression in patients. We studied two LEDGF/p75 exonic variants I436S and T473I, identified in HIV-1 long-term non-progressors, together with Q472L identified(mainly) in patients from South African cohorts. In vitro binding affinities of wild type, I436S, T473I, and Q472L LEDGF/p75 for HIV-1 integrase were comparable. When HIV-1 replication was evaluated in human somaticLEDGF/p75 knockout cells and LEDGF/p75 knockdown cells, complemented with wild type LEDGF/p75 or LEDGF/p75 variants, all variants rescued HIV-1replication to the same extent as wild type LEDGF/p75 whereas LEDGF/p75D366N, defective for interaction with HIV-1 integrase, did not. Our study indicated that mutations I436S and T473I did not alter the interaction of LEDGF/p75 with HIV-1 integrase, although identified in a cohort of long-term non-progressors, but validate our system to evaluate (future) variants observed in patients. Our conclusions can be summarized as follows:· A human cancer cell line knockout for LEDGF/p75 is viable.· In the absence of LEDGF/p75, residual HIV-1 replication can be observed, but only using laboratory HIV-1 strains.· In the absenceof LEDGF/p75, HRP-2 mediates HIV-1 replication. In wild type cells LEDGF/p75 is the dominant cellular co-factor.· LEDGINs are active in theabsence of LEDGF/p75 and inhibit the interaction of HIV-1 integrase with both the IBD of LEDGF/p75 and HRP-2.· LEDGF/p75 depletion shifts the HIV-1 integration site distribution more towards random, consistent with previous results. In the absence of LEDGF/p75, HIV-1 integration remains distinct from random, which can be explained in part by residual targeting via HRP-2.· LEDGF/p75 knockout affects both early and late steps of the HIV-1 replication cycle, the latter through a hitherto unknown mechanism.· LEDGF/p75 KO cell lines provide the means to study the effect of LEDGF/p75 variants observed in patients (e.g. I436S, Q472L, T473I). Future perspectivesThis study however left some questions unanswered and as research progresses, new questions have risen. Serial passaging of HIV-1 on cells lacking LEDGF/p75 led to the emergence of HIV containing integrase with the mutation A128T. Although this mutation did not seem to significantly increase viral replication capacity in LEDGF/p75 knockout cells, its retrieval is intriguing since A128T also confers resistance to first-generation LEDGINs. Next, the role of LEDGF/p75 during the late steps of viral replication remains to be elucidated. Our results indicate that virions produced in the absence ofLEDGF/p75 are impaired in the replication steps following entry in the next host cell. In the thesis dissertation of Drs Belete A Desimmie, we demonstrate that LEDGF/p75 is incorporated in the viral particle, suggesting that LEDGF/p75 is transferred by the virus and might play a role during subsequent early steps in the next cell. However, the mechanism andreasons why this event would take place requires further research. HIV integration is a crucial step during viral replication that archives the genetic material into the host cell DNA, enabling both a productive infection as well as the formation of a latent viral reservoir. The presence of such a reservoir necessitates life-long treatment with antiretroviral therapy as it enables the virus to evade the immune system or to rebound once antiretroviral therapy is withdrawn. The transcriptional status of HIV is tightly coupled to the activation state of its host cell. For instance in resting CD4+ T-cells studies indicate that chromatin remodelling induces silencing, a process which can be alleviated uponactivation of the cell. Next, integration itself could occasionally occur in a less favourable chromatin environment, thereby generating a latent provirus. Previous and current work demonstrates that LEDGF/p75 is the major targeting factor for HIV-1 integration. The effect of retargeting integration on the reactivation potential of HIV, however, has not been studied in depth. In line with this targeting role for LEDGF/p75, chimeras carrying alternative chromatin binding motifs fused to IBD could retarget HIV-1 integration. Our model provides the means to conceptually study the question if and how retargeting affects the reactivation potential. Since the AIDS pandemic is largely attributable to HIV-1, much less research has focused on HIV-2. Nevertheless the sharp contrastin clinical course between HIV-1 and HIV-2 infected patients (leading to progressive disease or AIDS in roughly 95% versus 20% of cases respectively, before the advent of antiretroviral therapy) merits further investigation of HIV-2. Intriguingly, there are clues that the proviral load of HIV-2 and HIV-1 are comparable, yet the cellular RNA levels and corresponding plasma viral load are significantly lower in HIV-2. The reason for this remains unknown and whether this is a consequence rather than acause of the different clinical course is unknown. One (of many!) possible explanations is that the integration pattern of HIV-2 differs from HIV-1. The HIV-1 integration pattern is strongly influenced by LEDGF/p75,but HIV-1 and HIV-2 integrase differ at crucial LEDGF/p75-interacting amino acids. Being a LEDGF/p75 look-a-like, HRP-2 contains a similarIBD but is not a dominant co-factor or targeting factor in HIV-1. We hypothesized that differential binding of HIV-1 and HIV-2 integrase to LEDGF/p75and/or HRP-2 might affect the integration site distribution. This comparative study is ongoing.The work presented in this dissertation alsotranslates beyond HIV. Our cell model enables the further characterization of the cellular role(s) of LEDGF/p75. Our model can be applied to study the effect of LEDGF/p75 alone on gene expression, since our knockoutleaves the LEDGF/p52 splice variant unaltered. This is an important distinction from previous Psip1 knockout models in mouse cells in which both splice variants, both suggested to act as a transcriptional co-activator, were knocked out. Recently, LEDGF/p75 was suggested to play a role in the DNA repair machinery for double strand breaks via the homologous recombination pathway. Again this was studied using RNAi or in Psip1 knockout mouse cells. Our cell model would enable the study of a specific LEDGF/p75 effect in a human context. A cancer cell line model inherently carries the problem of potential selection artefacts and not always translates to physiological conditions, however, remains a valid system for the initial study of cellular processes when these shortcomings are takencorrectly into consideration.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Biochemistry, Kulak (-)
Laboratory of Clinical Immunology
Molecular Virology and Gene Therapy
Faculty of Medicine, Campus Kulak Kortrijk

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