Hassen S. Wollebo

Persistence and pathogenesis of the neurotropic polyomavirus JC.

Many neurological diseases of the central nervous system (CNS) are underpinned by malfunctions of the immune system, including disorders involving opportunistic infections. Progressive multifocal leukoencephalopathy (PML) is a lethal CNS demyelinating disease caused by the human neurotropic polyomavirus JC (JCV) and is found almost exclusively in individuals with immune disruption, including patients with human immunodeficiency virus/acquired immunodeficiency syndrome, patients receiving therapeutic immunomodulatory monoclonal antibodies to treat conditions such as multiple sclerosis, and transplant recipients. Thus, the public health significance of this disease is high, because of the number of individuals constituting the at‐risk population. The incidence of PML is very low, whereas seroprevalence for the virus is high, suggesting infection by the virus is very common, and so it is thought that the virus is restrained but it persists in an asymptomatic state that can only occasionally be disrupted to lead to viral reactivation and PML. When JCV actively replicates in oligodendrocytes and astrocytes of the CNS, it produces cytolysis, leading to formation of demyelinated lesions with devastating consequences. Defining the molecular nature of persistence and events leading to reactivation of the virus to cause PML has proved to be elusive. In this review, we examine the current state of knowledge of the JCV life cycle and mechanisms of pathogenesis. We will discuss the normal course of the JCV life cycle including transmission, primary infection, viremia, and establishment of asymptomatic persistence as well as pathogenic events including migration of the virus to the brain, reactivation from persistence, viral infection, and replication in the glial cells of the CNS and escape from immunosurveillance. Ann Neurol 2015;77:560–570

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.

Role for tumor necrosis factor-alpha in JC virus reactivation and progressive multifocal leukoencephalopathy

JCV causes the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). After primary infection, JCV persists in a latent state, where viral protein expression and replication are not detectable. NF-κB and C/EBPβ regulate the JCV promoter via a control element, κB, suggesting proinflammatory cytokines may reactivate JCV to cause PML, e.g., in HIV-1/AIDS. Since HIV-1 induces cytokines in brain, including TNF-α, we examined a role for TNF-α in JCV regulation. TNF-α stimulated both early and late JCV transcription. Further, the κB element conferred TNF-α response to a heterologous promoter. Immunohistochemistry of HIV+/PML revealed robust labeling for TNF-α and TNFR-1. These data suggest TNF-α stimulation of κB may contribute to JCV reactivation in HIV+/PML.

Modulation of JC virus transcription by C/EBPβ

The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-kappaB binding site previously shown to activate transcription. We now report that C/EBPbeta inhibits basal and NF-kappaB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPbeta bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-kappaB/p65, C/EBPbeta-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPbeta-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPbeta negatively regulates JCV, which together with NF-kappaB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.

Inhibition of HSV-1 Replication by Gene Editing Strategy

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Zika virus: An emergent neuropathological agent.

The emergence of Zika virus in the Americas has followed a pattern that is familiar from earlier epidemics of other viruses, where a new disease is introduced into a human population and then spreads rapidly with important public health consequences. In the case of Zika virus, an accumulating body of recent evidence implicates the virus in the etiology of serious pathologies of the human nervous system, that is, the occurrence of microcephaly in neonates and Guillain–Barré syndrome in adults. Zika virus is an arbovirus (arthropod‐borne virus) and a member of the family Flaviviridae, genus Flavivirus. Zika virions are enveloped and icosahedral, and contain a nonsegmented, single‐stranded, positive‐sense RNA genome, which encodes 3 structural and 7 nonstructural proteins that are expressed as a single polyprotein that undergoes cleavage. Zika genomic RNA replicates in the cytoplasm of infected host cells. Zika virus was first detected in 1947 in the blood of a febrile monkey in Ugandas Zika Forest and in crushed suspensions of the Aedes mosquito, which is one of the vectors for Zika virus. The virus remained obscure, with a few human cases confined to Africa and Asia. There are two lineages of the Zika virus, African and Asian, with the Asian strain causing outbreaks in Micronesia in 2007 and French Polynesia in 2013–2014. From here, the virus spread to Brazil with the first report of autochthonous Zika transmission in the Americas in March 2015. The rapid advance of the virus in the Americas and its likely association with microcephaly and Guillain–Barré syndrome make Zika an urgent public health concern. Ann Neurol 2016;80:479–489

Cooperative roles of NF-κB and NFAT4 in polyomavirus JC regulation at the KB control element

The human polyomavirus JC (JCV) is the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is extremely common and after primary infection, JCV persists in a latent state. However, PML is a very rare disease suggesting that the virus is tightly regulated. Previously, we showed that NF-κB and C/EBPβ regulate the JCV early and late promoters via a DNA control element, KB, which also mediates the stimulatory effects of proinflammatory cytokines such as TNF-α on JCV gene expression. Other studies have implicated NFAT4 in JCV regulation. We now report that NFAT4 and NF-κB interact at the KB element to co-operatively activate both JCV early and late transcription and viral DNA replication. This interplay is inhibited by C/EBPβ and by agents that block the calcineurin/NFAT signaling pathway. The importance of these events in the regulation of JCV latency and reactivation is discussed.

Epigenetic regulation of polyomavirus JC

BackgroundPolyomavirus JC (JCV) causes the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), which occurs almost exclusively in people with immune deficiencies, such as HIV-1/AIDS patients. JCV infection is very common and usually occurs early in life. After primary infection, virus is controlled by the immune system but, rarely when immune function is impaired, it can re-emerge and multiply in the astrocytes and oligodendrocytes in the brain and cause PML. Thus a central question in PML pathogenesis is the nature of the molecular mechanisms maintaining JCV in a latent state and then allowing reactivation.MethodsSince transcription can be regulated by epigenetic mechanisms including DNA methylation and histone acetylation, we investigated their role in JCV regulation by employing inhibitors of epigenetic events.ResultsThe histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate powerfully stimulated JCV early and late transcription while the DNA methylation inhibitor 5-azacytidine had no effect. Analysis of JCV mutants showed that this effect was mediated by the KB element of the JCV control region, which binds transcription factors NF-κB p65, NFAT4 and C/EBPβ and mediates stimulation by TNF-α. Stimulation of transcription by p65 was additive with TSA as was cotransfection with transcriptional coactivators/acetyltransferase p300 whereas depletion of endogenous p65 by RNA interference inhibited the effect of TSA. EMSA with a KB oligonucleotide showed p65 expression, TNF-α stimulation or TSA treatment each caused a gel shift that was further shifted by antibody to p65.ConclusionsWe conclude that JCV is regulated epigenetically by protein acetylation events and that these involve the NF-κB p65 binding site in the JCV control region.

Expression of Signaling Molecules in Progressive Multifocal Leukoencephalopathy

INTRODUCTION Progressive multifocal leukoencephalopathy (PML) is a debilitating demyelinating disease of the CNS caused by the infection and destruction of glial cells by JC virus (JCV) and is an AIDS-defining disease. Infection with JCV is common and most people acquire antibodies early in life. After initial infection, JCV remains in an asymptomatic persistent state and can be detected by PCR in many tissues including brain. A major question in PML pathogenesis is how the virus reactivates from persistence in HIV-1/AIDS. Our studies with primary cultures of glial cells have implicated transcription factors NF-κB and NFAT4, which bind to a unique site in the JCV noncoding control region and stimulate viral gene expression. Furthermore, these transcription factors are controlled by pathways downstream of proinflammatory cytokines, e.g., TNF-α activates NF-κB and stimulates JCV transcription. OBJECTIVES We hypothesize that HIV-1/PML initiation may involve reactivation of JCV by cytokine disturbances in the brain such as occur in HIV-1/AIDS. In this study, the objective was to evaluate HIV-1/PML clinical samples for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 compared to non-PML controls. METHODS We evaluated HIV-1/PML clinical samples and non-PML controls for expression of TNF-α and its receptors and subcellular localization of NF-κB p65 and NFAT4 using Western blot and immunohistochemistry. RESULTS Consistent with our hypothesis, compared to non-PML controls, HIV-1/PML tissue has high levels of TNF-α and TNFR1 expression and NF-κB and NFAT4 were preferentially localized to the nucleus. CONCLUSION The involvement of TNF-α/NF-κB/NFAT4 signaling in JCV regulation that we reported from experiments in cultured human glial cells may be clinically relevant in PML.

The Brd4 acetyllysine-binding protein is involved in activation of polyomavirus JC

Brd4 is an epigenetic reader protein and a member of the BET (bromodomain and extra terminal domain) family of proteins with two bromodomains that recognize acetylated lysine residues. Brd4 specifically binds to acetylated transcription factor NF-κB p65 and coactivates transcription. Polyomavirus JC (JCV) is regulated by a noncoding control region (NCCR) containing promoter/enhancer elements for viral gene expression including a binding site for NF-κB, which responds to proinflammatory cytokines such as TNF-α, the DNA damage response, calcium signaling and acetylation of the NF-κB p65 subunit on lysine residues K218 and K221. Earlier studies indicated that NF-κB is involved in the reactivation of persistent/latent JCV in glial cells to cause progressive multifocal leukoencephalopathy (PML), a severe demyelinating disease of the brain caused by replication of JCV in glial cells. To investigate the mechanism of action of NF-κB acetylation on JCV transcription, we examined Brd4 and found that JCV early transcription was stimulated by Brd4 via the JCV NF-κB site and that p65 K218 and K221 were involved. Treatment with the Brd4 inhibitor JQ1(+) or mutation of either K218 or K221 to glutamine (K218R or K221) inhibited this stimulation and decreased the proportion of p65 in the nucleus. We conclude that Brd4 is involved in the regulation of the activation status of JCV in glial cells.