SMARCB1 deletion in atypical teratoid rhabdoid tumors results in human endogenous retrovirus K (HML-2) expression.

Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of SMARCB1, a tumor suppressor gene. In this study, we found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated. SMARCB1 knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of SMARCB1 expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation, and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates MAPK/ERK signaling in AT/RT cells. Overexpression of NRAS was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of NRAS, was bound to the HERV-K LTR significantly more in the absence of SMARCB1 expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on SMARCB1 and its interaction with MYC.

Advances toward Curing HIV-1 Infection in Tissue Reservoirs.

A disease of more than 39.6 million people worldwide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts toward a sterilizing or functional cure. On the horizon is research concentrated in multiple separate but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including checkpoint inhibitors, and gene editing. Here, we review the concept of targeting the HIV-1 tissue reservoirs, with an emphasis on the central nervous system, and describe relevant new work in functional cure research and strategies for HIV-1 eradication.

Presence of Tat and transactivation response element in spinal fluid despite antiretroviral therapy.

OBJECTIVE: The aim of this study was to measure the protein concentration and biological activity of HIV-1 Tat in cerebrospinal fluid (CSF) of individuals on suppressive antiretroviral therapy (ART). DESIGN: CSF was collected from 68 HIV-positive individuals on ART with plasma viral load less than 40 copies/ml, and from 25 HIV-negative healthy controls. Duration of HIV infection ranged from 4 to more than 30 years. METHODS: Tat levels in CSF were evaluated by an ELISA. Tat protein and viral RNA were quantified from exosomes isolated from CSF, followed by western blot or quantitative reverse transcription PCR, respectively. Functional activity of Tat was assessed using an LTR transactivation assay. RESULTS: Tat protein was detected in 36.8% of CSF samples from HIV-positive patients. CSF Tat concentration increased in four out of five individuals after initiation of therapy, indicating that Tat was not inhibited by ART. Similarly, exosomes from 34.4% of CSF samples were strongly positive for Tat protein and/or TAR RNA. Exosomal Tat retained transactivation activity in a CEM-LTR reporter assay in 66.7% of samples assayed, which indicates that over half of the Tat present in CSF is functional. Presence of Tat in CSF was highly associated with previous abuse of psychostimulants (cocaine or amphetamines; P = 0.01) and worse performance in the psychomotor speed (P = 0.04) and information processing (P = 0.02) cognitive domains. CONCLUSION: Tat and TAR are produced in the central nervous system despite adequate ART and are packaged into CSF exosomes. Tat remains biologically active within this compartment. These studies suggest that Tat may be a quantifiable marker of the viral reservoir and highlight a need for new therapies that directly inhibit Tat.

Human immunodeficiency virus type 1 (HIV-1) transactivator of transcription through its intact core and cysteine-rich domains inhibits Wnt/ß-catenin signaling in astrocytes: relevance to HIV neuropathogenesis.

Wnt/ß-catenin is a neuroprotective pathway regulating cell fate commitment in the CNS and many vital functions of neurons and glia. Its dysregulation is linked to a number of neurodegenerative diseases. Wnt/ß-catenin is also a repressor of HIV transcription in multiple cell types, including astrocytes, which are dysregulated in HIV-associated neurocognitive disorder. Given that HIV proteins can overcome host restriction factors and that perturbations of Wnt/ß-catenin signaling can compromise astrocyte function, we evaluated the impact of HIV transactivator of transcription (Tat) on Wnt/ß-catenin signaling in astrocytes. HIV clade B Tat, in primary progenitor-derived astrocytes and U87MG cells, inhibited Wnt/ß-catenin signaling as demonstrated by its inhibition of active ß-catenin, TOPflash reporter activity, and Axin-2 (a downstream target of Wnt/ß-catenin signaling). Point mutations in either the core region (K41A) or the cysteine-rich region (C30G) of Tat abrogated its ability to inhibit ß-catenin signaling. Clade C Tat, which lacks the dicysteine motif, did not alter ß-catenin signaling, confirming that the dicysteine motif is critical for Tat inhibition of ß-catenin signaling. Tat coprecipitated with TCF-4 (a transcription factor that partners with ß-catenin), suggesting a physical interaction between these two proteins. Furthermore, knockdown of ß-catenin or TCF-4 enhanced docking of Tat at the TAR region of the HIV long terminal repeat. These findings highlight a bidirectional interference between Tat and Wnt/ß-catenin that negatively impacts their cognate target genes. The consequences of this interaction include alleviation of Wnt/ß-catenin-mediated suppression of HIV and possible astrocyte dysregulation contributing to HIV neuropathogenesis.

Identification of novel T cell factor 4 (TCF-4) binding sites on the HIV long terminal repeat which associate with TCF-4, ß-catenin, and SMAR1 to repress HIV transcription.

Molecular regulation of HIV transcription is a multifaceted process dictated in part by the abundance of cellular transcription factors that induce or repress HIV promoter activity. ß-Catenin partners with members of the T cell factor (TCF)/LEF transcription factors to regulate gene expression. The interaction between ß-catenin and TCF-4 is linked to inhibition of HIV replication in multiple cell types, including lymphocytes and astrocytes. Here, we evaluated the molecular mechanism by which ß-catenin/TCF-4 repress HIV replication. We identified for the first time multiple TCF-4 binding sites at -336, -143, +66, and +186 relative to the transcription initiation site on the HIV long terminal repeat (LTR). Two of the sites (-143 and +66) were present in approximately 1/3 of 500 HIV-1 isolates examined. Although all four sites could bind to TCF-4, the strongest association occurred at -143. Deletion and/or mutation of -143, in conjunction with ß-catenin or TCF-4 knockdown in cells stably expressing an LTR reporter construct, enhanced basal HIV promoter activity by 5-fold but had no effect on Tat-mediated transactivation of the HIV LTR. We also found that TCF-4, ß-catenin, and the nuclear matrix binding protein SMAR1 tether at the -143-nucleotide (nt) site on the HIV LTR to inhibit HIV promoter activity. Collectively, these data indicate that TCF-4 and ß-catenin at -143 associate with SMAR1, which likely pulls the HIV DNA segment into the nuclear matrix and away from transcriptional machinery, leading to repression of basal HIV LTR transcription. These studies point to novel avenues for regulation of HIV replication by manipulation of ß-catenin signaling within cells.

Role of ß-catenin and TCF/LEF family members in transcriptional activity of HIV in astrocytes.

The Wnt/ß-catenin pathway is involved in diverse cell functions governing development and disease. ß-Catenin, a central mediator of this pathway, binds to members of the TCF/LEF family of transcription factors to modulate hundreds of genes. Active Wnt/ß-catenin/TCF-4 signaling plays a significant role in repression of HIV-1 replication in multiple cell targets, including astrocytes. To determine the mechanism by which active ß-catenin/TCF-4 leads to inhibition of HIV replication, we knocked down ß-catenin or TCF/LEF members in primary astrocytes and astrocytomas transiently transfected with an HIV long terminal repeat (LTR)-luciferase reporter that contained an integrated copy of the HIV LTR-luciferase construct. Knockdown of either ß-catenin or TCF-4 induced LTR activity by 2- to 3-fold under both the episomal and integrated conditions. This knockdown also increased presence of serine 2-phosphorylated RNA polymerase II (Pol II) on the HIV LTR as well as enhanced its processivity. Knockdown of ß-catenin/TCF-4 also impacted tethering of other transcription factors on the HIV promoter. Specifically, knockdown of TCF-4 enhanced binding of C/EBPß, C/EBPδ, and NF-κB to the HIV LTR, while ß-catenin knockdown increased binding of C/EBPß and C/EBPδ but had no effect on NF-κB. Approximately 150 genes in astrocytes were impacted by ß-catenin knockdown, including genes involved in inflammation/immunity, uptake/transport, vesicular transport/exocytosis, apoptosis/cellular stress, and cytoskeleton/trafficking. These findings indicate that modulation of the ß-catenin/TCF-4 axis impacts the basal level of HIV transcription in astrocytes, which may drive low level/persistent HIV in astrocytes that can contribute to ongoing neuroinflammation, and this axis also has profound effects on astrocyte biology.

IFN-gamma mediates enhancement of HIV replication in astrocytes by inducing an antagonist of the beta-catenin pathway (DKK1) in a STAT 3-dependent manner.

Typically, IFN-γ is an antiviral cytokine that inhibits the replication of many viruses, including HIV. However, in the CNS, IFN-γ induces HIV-productive replication in astrocytes. Although astrocytes in vitro are refractory to HIV replication, recent in vivo evidence demonstrated that astrocytes are infected by HIV, and their degree of infection is correlated with proximity to activated macrophages/microglia. The ability of IFN-γ to induce HIV replication in astrocytes suggests that the environmental milieu is critical in regulating the permissiveness of astrocytes to HIV infection. We evaluated the mechanism by which IFN-γ relieves restricted HIV replication in astrocytes. We demonstrate that although astrocytes have robust endogenous ß-catenin signaling, a pathway that is a potent inhibitor of HIV replication, IFN-γ diminished ß-catenin signaling in astrocytes by 40%, as evaluated by both active ß-catenin protein expression and ß-catenin-mediated T cell factor/lymphoid enhancer reporter (TOPflash) activity. Further, IFN-γ-mediated inhibition of ß-catenin signaling was dependent on its ability to induce an antagonist of the ß-catenin signaling pathway, Dickkopf-related protein 1, in a STAT 3-dependent manner. Inhibition of STAT3 and Dickkopf-related protein 1 abrogated the ability of IFN-γ to enhance HIV replication in astrocytes. These data demonstrated that IFN-γ induces HIV replication in astrocytes by antagonizing the ß-catenin pathway. To our knowledge, this is the first report to point to an intricate cross-talk between IFN-γ signaling and ß-catenin signaling that may have biologic and virologic effects on HIV outcome in the CNS, as well as on broader processes where the two pathways interface.

Role of ß-catenin/TCF-4 signaling in HIV replication and pathogenesis: insights to informing novel anti-HIV molecular therapeutics.

A greater understanding of the interaction between HIV and host signaling pathways that restrict virus production may lead to new methods to purge virus from latent reservoirs and enhance survival/function of cells targeted by HIV. This review highlights the role of the Wnt/ß-catenin pathway as a host factor that represses HIV replication in multiple targets, especially those relevant to HIV in the central nervous system.