Cytomegalovirus and Cardiovascular Disease: A Hypothetical Role for Viral G-Protein-Coupled Receptors in Hypertension.

Cytomegalovirus (CMV) is a member of the ß-herpesviruses and is ubiquitous, infecting 50%-99% of the human population depending on ethnic and socioeconomic conditions. CMV establishes lifelong, latent infections in their host. Spontaneous reactivation of CMV is usually asymptomatic, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with several cardiovascular and post-transplant diseases (stroke, atherosclerosis, post-transplant vasculopathy, and hypertension). Herpesviruses, including CMV, encode viral G-protein-coupled receptors (vGPCRs) that alter the host cell by hijacking signaling pathways that play important roles in the viral life cycle and these cardiovascular diseases. In this brief review, we discuss the pharmacology and signaling properties of these vGPCRs, and their contribution to hypertension. Overall, these vGPCRs can be considered attractive targets moving forward in the development of novel hypertensive therapies.

Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused widespread morbidity and mortality since its onset in late 2019. Here, we demonstrate that prior infection with human cytomegalovirus (HCMV) substantially increases infection with SARS-CoV-2 in vitro. HCMV is a common herpesvirus carried by 40%-100% of the population, which can reactivate in the lung under inflammatory conditions, such as those resulting from SARS-CoV-2 infection. We show in both endothelial and epithelial cell types that HCMV infection upregulates ACE2, the SARS-CoV-2 cell entry receptor. These observations suggest that HCMV reactivation events in the lung of healthy HCMV carriers could exacerbate SARS-CoV-2 infection and subsequent COVID-19 symptoms. This effect could contribute to the disparity of disease severity seen in ethnic minorities and those with lower socioeconomic status, due to their higher CMV seroprevalence. Our results warrant further clinical investigation as to whether HCMV infection influences the pathogenesis of SARS-CoV-2.

IL-10-Secreting CD8+ T Cells Specific for Human Cytomegalovirus (HCMV): Generation, Maintenance and Phenotype.

HCMV-specific CD8+ T-cells are potent anti-viral effector cells in HCMV infected individuals, but evidence from other viral infections suggests that CD8+ T-cells can also produce the immunomodulatory cytokine IL-10. In this work we show that there are HCMV-specific IL-10 CD8+ T-cell responses in a cohort of individuals aged 23-76 years of age, predominantly directed against the HCMV proteins known to be expressed during latent infections as well as towards the proteins US3 and pp71. The analysis of HCMV-specific responses established during primary infection has shown that the IL-10 responses to US3 and pp71 HCMV proteins are detectable in the first weeks post infection, but not the responses to latency-associated proteins, and this IL-10 response is produced by both CD8+ and CD4+ T-cells. Phenotyping studies of HCMV-specific IL-10+ CD8+ T-cells show that these are CD45RA+ effector memory cells and co-express CD28 and CD57, however, the expression of the inhibitory receptor PD-1 varied from 90% to 30% between donors. In this study we have described for the first time the HCMV-specific IL-10 CD8+ T-cell responses and have demonstrated their broad specificity and the potential immune modulatory role of the immune response to HCMV latent carriage and periodic reactivation.

The Human Cytomegalovirus ß2.7 Long Non-Coding RNA Prevents Induction of Reactive Oxygen Species to Maintain Viral Gene Silencing during Latency.

Human cytomegalovirus (HCMV) is a significant source of disease for the immunosuppressed and immunonaive. The treatment of HCMV is made more problematic by viral latency, a lifecycle stage in which the virus reduces its own gene expression and produces no infectious virus. The most highly expressed viral gene during HCMV latency is the viral ß2.7 long non-coding RNA. Although we have recently shown that the ß2.7 lncRNA lowers levels of reactive oxygen species (ROS) during infection in monocytes, how this impacts latency is unclear. We now show that ß2.7 is important for establishing and maintaining HCMV latency by aiding the suppression of viral lytic gene expression and that this is directly related to its ability to quench reactive oxygen species (ROS). Consistent with this, we also find that exogenous inducers of ROS cause reactivation of latent HCMV. These effects can be compensated by treatment with an antioxidant to lower ROS levels. Finally, we show that ROS-mediated reactivation is independent of myeloid differentiation, but instead relies on NF-κB activation. Altogether, these results reveal a novel factor that is central to the complex process that underpins HCMV latency. These findings may be of particular relevance in the transplant setting, in which transplanted tissue/organs are subject to very high ROS levels, and HCMV reactivation poses a significant threat.

A Viral Long Non-Coding RNA Protects against Cell Death during Human Cytomegalovirus Infection of CD14+ Monocytes.

Long non-coding RNA ß2.7 is the most highly transcribed viral gene during latent human cytomegalovirus (HCMV) infection. However, as yet, no function has ever been ascribed to ß2.7 during HCMV latency. Here we show that ß2.7 protects against apoptosis induced by high levels of reactive oxygen species (ROS) in infected monocytes, which routinely support latent HCMV infection. Monocytes infected with a wild-type (WT) virus, but not virus deleted for the ß2.7 gene (Δß2.7), are protected against mitochondrial stress and subsequent apoptosis. Protected monocytes display lower levels of ROS and additionally, stress-induced death in the absence of ß2.7 can be reversed by an antioxidant which reduces ROS levels. Furthermore, we show that infection with WT but not Δß2.7 virus results in strong upregulation of a cellular antioxidant enzyme, superoxide dismutase 2 (SOD2) in CD14+ monocytes. These observations identify a role for the ß2.7 viral transcript, the most abundantly expressed viral RNA during latency but for which no latency-associated function has ever been ascribed, and demonstrate a novel way in which HCMV protects infected monocytes from pro-death signals to optimise latent carriage.

Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies.

Latent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

HCMV Antivirals and Strategies to Target the Latent Reservoir.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. In healthy people, primary infection is generally asymptomatic, and the virus can go on to establish lifelong latency in cells of the myeloid lineage. However, HCMV often causes severe disease in the immunosuppressed: transplant recipients and people living with AIDS, and also in the immunonaive foetus. At present, there are several antiviral drugs licensed to control HCMV disease. However, these are all faced with problems of poor bioavailability, toxicity and rapidly emerging viral resistance. Furthermore, none of them are capable of fully clearing the virus from the host, as they do not target latent infection. Consequently, reactivation from latency is a significant source of disease, and there remains an unmet need for treatments that also target latent infection. This review briefly summarises the most common HCMV antivirals used in clinic at present and discusses current research into targeting the latent HCMV reservoir.

Latent Cytomegalovirus-Driven Recruitment of Activated CD4+ T Cells Promotes Virus Reactivation.

Human cytomegalovirus (HCMV) infection is not cleared by the initial immune response but persists for the lifetime of the host, in part due to its ability to establish a latent infection in cells of the myeloid lineage. HCMV has been shown to manipulate the secretion of cellular proteins during both lytic and latent infection; with changes caused by latent infection mainly investigated in CD34+ progenitor cells. Whilst CD34+ cells are generally bone marrow resident, their derivative CD14+ monocytes migrate to the periphery where they briefly circulate until extravasation into tissue sites. We have analyzed the effect of HCMV latent infection on the secretome of CD14+ monocytes, identifying an upregulation of both CCL8 and CXCL10 chemokines in the CD14+ latency-associated secretome. Unlike CD34+ cells, the CD14+ latency-associated secretome did not induce migration of resting immune cell subsets but did induce migration of activated NK and T cells expressing CXCR3 in a CXCL10 dependent manner. As reported in CD34+ latent infection, the CD14+ latency-associated secretome also suppressed the anti-viral activity of stimulated CD4+ T cells. Surprisingly, however, co-culture of activated autologous CD4+ T cells with latently infected monocytes resulted in reactivation of HCMV at levels comparable to those observed using M-CSF and IL-1ß cytokines. We propose that these events represent a potential strategy to enable HCMV reactivation and local dissemination of the virus at peripheral tissue sites.

Viral G Protein-Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases.

Herpesviruses are ubiquitous pathogens that establish lifelong, latent infections in their host. Spontaneous reactivation of herpesviruses is often asymptomatic or clinically manageable in healthy individuals, but reactivation events in immunocompromised or immunosuppressed individuals can lead to severe morbidity and mortality. Moreover, herpesvirus infections have been associated with multiple proliferative cardiovascular and post-transplant diseases. Herpesviruses encode viral G protein-coupled receptors (vGPCRs) that alter the host cell by hijacking cellular pathways and play important roles in the viral life cycle and these different disease settings. In this review, we discuss the pharmacological and signaling properties of these vGPCRs, their role in the viral life cycle, and their contribution in different diseases. Because of their prominent role, vGPCRs have emerged as promising drug targets, and the potential of vGPCR-targeting therapeutics is being explored. Overall, these vGPCRs can be considered as attractive targets moving forward in the development of antiviral, cancer, and/or cardiovascular disease treatments. SIGNIFICANCE STATEMENT: In the last decade, herpesvirus-encoded G protein-coupled receptors (GPCRs) have emerged as interesting drug targets with the growing understanding of their critical role in the viral life cycle and in different disease settings. This review presents the pharmacological properties of these viral receptors, their role in the viral life cycle and different diseases, and the emergence of therapeutics targeting viral GPCRs.

Bromodomain proteins regulate human cytomegalovirus latency and reactivation allowing epigenetic therapeutic intervention.

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.

Bromodomain Inhibitors as Therapeutics for Herpesvirus-Related Disease: All BETs Are Off?

Although the ubiquitous human herpesviruses (HHVs) are rarely associated with serious disease of the healthy host, primary infection and reactivation in immunocompromised individuals can lead to significant morbidity and, in some cases, mortality. Effective drugs are available for clinical treatment, however resistance is on the rise such that new anti-viral targets, as well as novel clinical treatment strategies, are required. A promising area of development and pre-clinical research is that of inhibitors of epigenetic modifying proteins that control both cellular functions and the viral life cycle. Here, we briefly outline the interaction of the host bromo- and extra-terminal domain (BET) proteins during different stages of the HHVs' life cycles while giving a full overview of the published work using BET bromodomain inhibitors (BRDis) during HHV infections. Furthermore, we provide evidence that small molecule inhibitors targeting the host BET proteins, and BRD4 in particular, have the potential for therapeutic intervention of HHV-associated disease.

Human cytomegalovirus major immediate early transcripts arise predominantly from the canonical major immediate early promoter in reactivating progenitor-derived dendritic cells.

Human cytomegalovirus latency and reactivation is a major source of morbidity in immune-suppressed patient populations. Lifelong latent infections are established in CD34+progenitor cells in the bone marrow, which are hallmarked by a lack of major lytic gene expression, genome replication and virus production. A number of studies have shown that inhibition of the major immediate early promoter (MIEP) - the promoter that regulates immediate early (IE) gene expression - is important for the establishment of latency and that, by extension, reactivation requires reversal of this repression of the MIEP. The identification of novel promoters (termed ip1 and ip2) downstream of the MIEP that can drive IE gene expression has led to speculation over the precise role of the MIEP in reactivation. In this study we show that IE transcripts arise from both the MIEP and ip2 promoter in the THP1 cell macrophage cell line and also CD14+monocytes stimulated with phorbol ester. In contrast, we show that in in vitro generated dendritic cells or macrophages that support HCMV reactivation IE transcripts arise predominantly from the MIEP and not the intronic promoters. Furthermore, inhibition of histone modifying enzyme activity confirms the view that the MIEP is predominantly regulated by the activity of cellular chromatin. Finally, we observe that ip2-derived IE transcription is cycloheximide-sensitive in reactivating DCs, behaviour consistent with an early gene designation. Taken together, these data argue that MIEP activity is still important for HCMV reactivation but ip2 activity could play cell-type-specific roles in reactivation.

A Virally Encoded DeSUMOylase Activity Is Required for Cytomegalovirus Reactivation from Latency.

A subset of viral genes is required for the long-term latent infection of hematopoietic cells by human cytomegalovirus (HCMV). Here, we show that a latency-associated gene product (LUNA) promotes the disruption of cellular PML bodies during latency. Mutation and inhibitor studies reveal that LUNA encodes a deSUMOylase activity responsible for this disruption. Specifically, LUNA encodes a conserved Asp-Cys-Gly motif common to all deSUMOylases. Importantly, mutation of the putative catalytic cysteine is sufficient to reverse LUNA-mediated PML dispersal and markedly reduces the efficiency of viral reactivation. The depletion of PML from cells is sufficient to rescue the reactivation of the LUNA-deficient viruses, arguing that targeting PML is an important biological role of LUNA. Finally, we demonstrate that reactivation of naturally latent HCMV is blocked by deSUMOylase inhibitors. Thus, latent HCMV primes the cellular environment for efficient reactivation via the activity of a virally encoded deSUMOylase.

Latency-Associated Expression of Human Cytomegalovirus US28 Attenuates Cell Signaling Pathways To Maintain Latent Infection.

Reactivation of human cytomegalovirus (HCMV) latent infection from early myeloid lineage cells constitutes a threat to immunocompromised or immune-suppressed individuals. Consequently, understanding the control of latency and reactivation to allow targeting and killing of latently infected cells could have far-reaching clinical benefits. US28 is one of the few viral genes that is expressed during latency and encodes a cell surface G protein-coupled receptor (GPCR), which, during lytic infection, is a constitutive cell-signaling activator. Here we now show that in monocytes, which are recognized sites of HCMV latency in vivo, US28 attenuates multiple cell signaling pathways, including mitogen-activated protein (MAP) kinase and NF-κB, and that this is required to establish a latent infection; viruses deleted for US28 initiate a lytic infection in infected monocytes. We also show that these monocytes then become potent targets for the HCMV-specific host immune response and that latently infected cells treated with an inverse agonist of US28 also reactivate lytic infection and similarly become immune targets. Consequently, we suggest that the use of inhibitors of US28 could be a novel immunotherapeutic strategy to reactivate the latent viral reservoir, allowing it to be targeted by preexisting HCMV-specific T cells.IMPORTANCE Human cytomegalovirus (HCMV) is a betaherpesvirus and a leading cause of morbidity and mortality among immunosuppressed individuals. HCMV can establish latent infection, where the viral genome is maintained in an infected cell, without production of infectious virus. A number of genes, including US28, are expressed by HCMV during latent infection. US28 has been shown to activate many cellular signaling pathways during lytic infection, promoting lytic gene expression and virus production. As such, the role of US28 remains unclear and seems at odds with latency. Here, we show that US28 has the opposite phenotype in cells that support latent infection-it attenuates cellular signaling, thereby maintaining latency. Inhibition of US28 with a small-molecule inhibitor causes HCMV latent infection to reactivate, allowing latently infected cells to be detected and killed by the immune system. This approach could be used to treat latent HCMV to clear it from human transplants.

Latent Cytomegalovirus (CMV) Infection Does Not Detrimentally Alter T Cell Responses in the Healthy Old, But Increased Latent CMV Carriage Is Related to Expanded CMV-Specific T Cells.

Human cytomegalovirus (HCMV) primary infection and periodic reactivation of latent virus is generally well controlled by T-cell responses in healthy people. In older donors, overt HCMV disease is not generally seen despite the association of HCMV infection with increased risk of mortality. However, increases in HCMV DNA in urine of older people suggest that, although the immune response retains functionality, immunomodulation of the immune response due to lifelong viral carriage may alter its efficacy. Viral transcription is limited during latency to a handful of viral genes and there is both an IFNγ and cellular IL-10 CD4+ T-cell response to HCMV latency-associated proteins. Production of cIL-10 by HCMV-specific CD4+ T-cells is a candidate for aging-related immunomodulation. To address whether long-term carriage of HCMV changes the balance of cIL-10 and IFNγ-secreting T-cell populations, we recruited a large donor cohort aged 23-78 years and correlated T-cell responses to 11 HCMV proteins with age, HCMV IgG levels, latent HCMV load in CD14+ monocytes, and T-cell numbers in the blood. IFNγ responses by CD4+ and CD8+ T-cells to all HCMV proteins were detected, with no age-related increase in this cohort. IL-10-secreting CD4+ T cell responses were predominant to latency-associated proteins but did not increase with age. Quantification of HCMV genomes in CD14+ monocytes, a known site of latent HCMV carriage, did not reveal any increase in viral genome copies in older donors. Importantly, there was a significant positive correlation between the latent viral genome copy number and the breadth and magnitude of the IFNγ T-cell response to HCMV proteins. This study suggests in healthy aged donors that HCMV-specific changes in the T cell compartment were not affected by age and were effective, as viremia was a very rare event. Evidence from studies of unwell aged has shown HCMV to be an important comorbidity factor, surveillance of latent HCMV load and low-level viremia in blood and body fluids, alongside typical immunological measures and assessment of the antiviral capacity of the HCMV-specific immune cell function would be informative in determining if antiviral treatment of HCMV replication in the old maybe beneficial.

The immunology of human cytomegalovirus latency: could latent infection be cleared by novel immunotherapeutic strategies?

While the host immune response following primary human cytomegalovirus (HCMV) infection is generally effective at stopping virus replication and dissemination, virus is never cleared by the host and like all herpesviruses, persists for life. At least in part, this persistence is known to be facilitated by the ability of HCMV to establish latency in myeloid cells in which infection is essentially silent with, importantly, a total lack of new virus production. However, although the viral transcription programme during latency is much suppressed, a number of viral genes are expressed during latent infection at the protein level and many of these have been shown to have profound effects on the latent cell and its environment. Intriguingly, many of these latency-associated genes are also expressed during lytic infection. Therefore, why the same potent host immune responses generated during lytic infection to these viral gene products are not recognized during latency, thereby allowing clearance of latently infected cells, is far from clear. Reactivation from latency is also a major cause of HCMV-mediated disease, particularly in the immune compromised and immune naive, and is also likely to be a major source of virus in chronic subclinical HCMV infection which has been suggested to be associated with long-term diseases such as atherosclerosis and some neoplasias. Consequently, understanding latency and why latently infected cells appear to be immunoprivileged is crucial for an understanding of the pathogenesis of HCMV and may help to design strategies to eliminate latent virus reservoirs, at least in certain clinical settings.

The use of primary human cells (fibroblasts, monocytes, and others) to assess human cytomegalovirus function.

The extensive tropism of human cytomegalovirus (HCMV) results in the productive infection of multiple cell types within the human host. However, infection of other cell types, such as undifferentiated cells of the myeloid lineage, gives rise to nonpermissive infections. This has been used experimentally to model latent infection which is known to be established in the pluripotent CD34+ hematopoietic progenitor cell population resident in the bone marrow in vivo. The absence of a tractable animal model for studies of HCMV has resulted in a number of laboratories employing experimental infection of cells in vitro to simulate both HCMV lytic and latent infection. Herein, we will focus on the techniques used in our laboratory for the isolation and use of primary cells to study aspects of HCMV latency, reactivation, and lytic infection.

Human cytomegalovirus manipulation of latently infected cells.

Primary infection with human cytomegalovirus (HCMV) results in the establishment of a lifelong infection of the host which is aided by the ability of HCMV to undergo a latent infection. One site of HCMV latency in vivo is in haematopoietic progenitor cells, resident in the bone marrow, with genome carriage and reactivation being restricted to the cells of the myeloid lineage. Until recently, HCMV latency has been considered to be relatively quiescent with the virus being maintained essentially as a "silent partner" until conditions are met that trigger reactivation. However, advances in techniques to study global changes in gene expression have begun to show that HCMV latency is a highly active process which involves expression of specific latency-associated viral gene products which orchestrate major changes in the latently infected cell. These changes are argued to help maintain latent infection and to modulate the cellular environment to the benefit of latent virus. In this review, we will discuss these new findings and how they impact not only on our understanding of the biology of HCMV latency but also how they could provide tantalising glimpses into mechanisms that could become targets for the clearance of latent HCMV.

Circulating dendritic cells isolated from healthy seropositive donors are sites of human cytomegalovirus reactivation in vivo.

Primary infection with human cytomegalovirus (HCMV) is generally asymptomatic in healthy individuals and results in a lifelong infection of the host. In contrast, in immunosuppressed transplant recipients and late-stage AIDS patients, HCMV infection and reactivation can result in severe disease or death. In vivo, latency is established in bone marrow CD34(+) progenitor cells with reactivation linked with their differentiation to macrophages and dendritic cells (DCs). However, previous analyses have relied on ex vivo differentiation of myeloid progenitor cells to DCs in culture. Here, we now report on the isolation and analysis of circulating blood myeloid DCs, resulting from natural differentiation in vivo, from healthy HCMV-seropositive carriers. We show that these in vivo-differentiated circulating DCs are fully permissive for HCMV and exhibit a phenotype similar to that of monocyte-derived DCs routinely used for in vitro studies of HCMV. Importantly, we also show that these DCs from healthy HCMV-seropositive donors carry HCMV genomes and, significantly, are typically positive for viral immediate-early (IE) gene expression, in contrast to circulating monocytes, which carry genomes with an absence of IE expression. Finally, we show that HCMV reactivation from these circulating DCs is enhanced by inflammatory stimuli. Overall, these data argue that the differentiation in vivo of myeloid progenitors to circulating DCs promotes the reactivation of HCMV lytic gene expression in healthy individuals, thereby providing valuable confirmation of studies performed using in vitro generation of DCs from myeloid precursors to study HCMV reactivation.

Latency-associated degradation of the MRP1 drug transporter during latent human cytomegalovirus infection.

The reactivation of latent human cytomegalovirus (HCMV) infection after transplantation is associated with high morbidity and mortality. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, whose establishment and/or maintenance require expression of the viral transcript UL138. Using stable isotope labeling by amino acids in cell culture-based mass spectrometry, we found a dramatic UL138-mediated loss of cell surface multidrug resistance-associated protein-1 (MRP1) and the reduction of substrate export by this transporter. Latency-associated loss of MRP1 and accumulation of the cytotoxic drug vincristine, an MRP1 substrate, depleted virus from naturally latent CD14(+) and CD34(+) progenitors, all of which are in vivo sites of latency. The UL138-mediated loss of MRP1 provides a marker for detecting latent HCMV infection and a therapeutic target for eliminating latently infected cells before transplantation.