PNAS Article: Epigenetic Targeting of HCMV Latency

Significance

Human cytomegalovirus (HCMV) reactivation is a major cause of posttransplant morbidity/mortality. One approach toward reducing this is purging the transplant donor and/or recipient of latently infected cells prior to stem-cell or organ harvest/engraftment. Our findings show the involvement of host bromodomain (BRD) proteins in regulation of HCMV latency and reactivation. Bromodomain and extra-terminal inhibitor (I-BET) treatment of latently infected cells causes reactivation of lytic gene expression by release of transcription activator P-TEFb (CDK9/CycT1) from BRD4-associated repressive complexes, with subsequent recruitment via host superelongation complex (SEC). This results in immune targeting and T cell-mediated killing of these otherwise latently infected cells and provides a therapeutic “shock and kill” strategy that could reduce HCMV-mediated disease in the transplant setting.

Abstract

Reactivation of human cytomegalovirus (HCMV) from latency is a major health consideration for recipients of stem-cell and solid organ transplantations. With over 200,000 transplants taking place globally per annum, virus reactivation can occur in more than 50% of cases leading to loss of grafts as well as serious morbidity and even mortality. Here, we present the most extensive screening to date of epigenetic inhibitors on HCMV latently infected cells and find that histone deacetylase inhibitors (HDACis) and bromodomain inhibitors are broadly effective at inducing virus immediate early gene expression. However, while HDACis, such as myeloid-selective CHR-4487, lead to production of infectious virions, inhibitors of bromodomain (BRD) and extraterminal proteins (I-BETs), including GSK726, restrict full reactivation. Mechanistically, we show that BET proteins (BRDs) are pivotally connected to regulation of HCMV latency and reactivation. Through BRD4 interaction, the transcriptional activator complex P-TEFb (CDK9/CycT1) is sequestered by repressive complexes during HCMV latency. Consequently, I-BETs allow release of P-TEFb and subsequent recruitment to promoters via the superelongation complex (SEC), inducing transcription of HCMV lytic genes encoding immunogenic antigens from otherwise latently infected cells. Surprisingly, this occurs without inducing many viral immunoevasins and, importantly, while also restricting viral DNA replication and full HCMV reactivation. Therefore, this pattern of HCMV transcriptional dysregulation allows effective cytotoxic immune targeting and killing of latently infected cells, thus reducing the latent virus genome load. This approach could be safely used to pre-emptively purge the virus latent reservoir prior to transplantation, thereby reducing HCMV reactivation-related morbidity and mortality.

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Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that infects between 50 and 90% of the world’s population. Primary infection with HCMV usually results in lifelong carriage of the virus in a latent state, in the absence of infectious virus production, within cells of the myeloid lineage. Subsequent virus reactivation events are usually asymptomatic in a normal healthy individual. However, severe disease is seen following HCMV reactivation in immunocompromised individuals, such as AIDS and transplant patients (1). Reactivation in the transplant recipient or reinfection via reactivation from the donor graft can lead to severe morbidity, including organ rejection and, in some cases, mortality (2). The serostatus of both donor and recipient leads to a graded risk of reactivation and clinical outcome during immunosuppression after solid organ transplantation (SOT), which is highest with seropositive donor to seronegative recipient (D+/R‒) (3). The rate of HCMV reactivation also varies depending on the type of transplantation, with allogeneic SOT and hematopoietic stem-cell transplantation (HSCT) having HCMV-associated disease as high as 56 and 80%, respectively, in highly immune-suppressed seropositive patients (4). Given that kidney, lung, and HSCT transplants alone now globally exceed 200,000 annually (www.transplant-observatory.org), a very high number of patients will experience HCMV reactivation events and potential HCMV-mediated disease unless effective intervention strategies can be devised.

While advancements have been made in designing a vaccine against HCMV, no candidate is currently effective (5). Additionally, while both prophylactic and pre-emptive antiviral therapies can be efficacious for many, for a significant number of patients these can be both ineffective and detrimental; despite improvements with the recently licensed letermovir, drugs such as (val)gancylcovir are poorly bioavailable and can involve high toxicity, resulting in neutropenia (6). Moreover, a major issue is the development of drug resistance, examples of which are already on the rise even with letermovir (7), while therapies such as adoptive T cell transfer can aggravate graft versus host disease with HCMV reactivation (3).

These issues have more recently led to investigations into the potential of directly targeting the latent reservoir; reducing the number of latently infected cells of both seropositive donors and recipients prior to transplantation could reduce the chances of HCMV reactivation posttransplant and provide a better clinical outcome (8). Studies have shown that targeting HCMV latently infected cells, namely myeloid lineage CD14+ monocytes and their CD34+ progenitors (9), can be employed; findings include HCMV-driven down-regulation of multidrug resistance protein-1, which allows specific killing of latent cells with the chemotherapy drug vincristine (10). Work from our group has also shown that targeting HCMV-specific proteins expressed on the surface of latently infected cells, such as US28, using fusion toxin proteins also allows killing of both experimentally and naturally latent HCMV-infected cells in vitro (11).

An alternative approach is to use immune-mediated killing of latently infected cells by enabling recognition through host T cells. While cells undergoing lytic infection can be recognized by naturally present host cytotoxic T cells (CTLs), HCMV latently infected cells do not efficiently present these lytic antigens (8). As such, drugs that induce latently infected cells to express viral lytic proteins should lead to recognition and elimination of latent cells by circulating HCMV-specific CTLs. Commonly, this has become known as “shock and kill” therapy and has led to various latency reversal agents (LRAs), including histone deacetylase inhibitors (HDACis), being trialled with HIV (12). Latency of HCMV in the myeloid lineage is maintained through chromatin-mediated epigenetic repression of the major immediate early promoter (MIEP) (13), with activation of the MIEP usually driven by differentiation of myeloid cells to macrophage or dendritic cells (DCs) (14). We have previously shown that, using HDACis such as valproic acid (VPA) and MC1568, transient HCMV lytic gene expression can be induced, which allows CTL recognition and killing of both experimentally and naturally latent infected cells (15). However, these particular HDACis only caused maximal induction of immediate early gene expression in around 25% of latently infected cells, and HDACis have been shown elsewhere to impair the activity of T cells when targeting HIV reactivation (16).

Newer-generation HDACis with higher potency and lower side effects are now available, as well as inhibitors to a variety of epigenetic modifying enzymes possibly having a role in regulating expression from the HCMV MIEP (17, 18). While targeting epigenetic enzymes in vitro, such as histone demethylases (HDMs), has resulted largely in decreases in HCMV gene expression and virus yield (19, 20), as well as restriction of replication and reactivation of other herpesviruses (21), inhibiting the histone methyltransferase (HMT) component of polycomb-repressive complex-2 (PRC2), EZH2, across two different studies has both activated lytic gene expression (22) and, conversely, suppressed infection by inducing an antiviral cellular state (23). With this in mind, we hypothesized that investigating a range of epigenetic inhibitors for their ability to target possible host regulators of HCMV lytic gene expression might cause increased numbers of latently infected cells to become visible to the host immune system without causing full virus reactivation and avoiding induction of HCMV immune evasion genes, which could protect latently infected cells from CTL recognition.

Here, we show that continuous treatment of latently infected cells with certain HDACis causes not just activation of HCMV gene expression but full reactivation and lytic cascade of the virus. In contrast, through the release of the cellular transcriptional activating complex positive-transcription elongation factor-b (P-TEFb) from repressive BRD4-associated aggregates, and recruitment via the superelongation complex (SEC) to viral promoters, inhibitors of the BET family of bromodomain (BRD)-containing histone acetylation mark reader proteins (I-BETs) (24, 25) induce dysregulated HCMV gene expression. This allows production of virus lytic immunogens, including immunodominant IE72, pp65, and gB, while restricting the expression of many viral immunomodulating proteins (e.g., MHC class I/II downmodulators US2, 3, 6, 8, and 11) and, most importantly, virus DNA replication machinery (e.g., UL44, UL112). Subsequently, these inhibitors allow efficient killing of both experimentally and naturally infected HCMV latent cells by host CTLs in the absence of virus productive infection and, ultimately, reduce the presence of HCMV viral DNA within ex-vivo–treated peripheral blood cells of HCMV seropositive individuals.

Results

BET Inhibitors Induce High MIEP Activity in Cell-Line Models of HCMV Latency.

The HDACi VPA is used for the clinical treatment of epilepsy and, in our previous study (15), we showed that treatment of HCMV latently infected ce