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The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Vpr enhances viral replication in both macrophages and in cycling T cells to a lesser extent. Virion packaged Vpr is released in target cells shortly after entry, suggesting its requirement in the early phase of infection. Previously, we described REAF (RNA-associated Early-stage Antiviral Factor, RPRD2), a constitutively expressed protein that potently restricts HIV replication at or during reverse transcription. Here, we show that a virus without intactvpris more highly restricted by REAF and, using delivery by VLPs, that Vpr alone is sufficient for REAF degradation in primary macrophages. REAF is more highly expressed in macrophages than in cycling T cells and we detect, by co-immunoprecipitation assay, an interaction between Vpr protein and endogenous REAF. Vpr acts very quickly during the early phase of replication and induces the degradation of REAF within 30 minutes of viral entry. Using Vpr F34I and Q65R viral mutants, we show that nuclear localisation and interaction with cullin4A-DBB1 (DCAF1) E3 ubiquitin ligase is required for REAF degradation by Vpr. In response to infection, cells upregulate REAF levels. This response is curtailed in the presence of Vpr. These findings support the hypothesis that Vpr induces the degradation of a factor, REAF, which impedes HIV infection in macrophages.ImportanceFor at least 30 years, it has been known that HIV-1 Vpr, a protein carried in the virion, is important for efficient infection of primary macrophages. Vpr is also a determinant of the pathogenic effects of HIV-1in vivo. A number of cellular proteins that interact with Vpr have been identified. So far, it has not been possible to associate these proteins with altered viral replication in macrophages, or to explain why Vpr is carried in the virus particle. Here we show that Vpr mitigates the antiviral effects of REAF, a protein highly expressed in primary macrophages and one which inhibits virus replication early during reverse transcription. REAF is degraded by Vpr within 30 minutes of virus entry, in a manner dependent on the nuclear localization of Vpr and its interaction with the cell’s protein degradation machinery.

The worldwide prevalence of diabetes has reached 8.5% among adults, and this is characterised by elevated glucose concentrations and failing insulin secretion. Furthermore, most people with type 2 diabetes are either obese or overweight, with the associated dyslipidaemia contributing to the development of insulin resistance and increased cardiovascular risk. Here we incubated INS-1 pancreatic β-cells for 72 h in RPMI-1640 media, or media supplemented with 28 mM glucose, 200 µM palmitic acid, and 200 µM oleic acid as a cellular model of diabetic glucolipotoxicity. Illumina HiSeq gene expression analysis showed the trace amine-associated receptor (TAAR) family to be among the most highly downregulated by glucolipotoxicity. Importantly, MetaCore integrated knowledge database, from Clarivate Analytics, indicated potential TAAR impact on insulin secretion through adenylyl cyclase signalling pathways. We therefore investigated the effect of TAAR ligands on cAMP signalling and insulin secretion, and found that only the branch of the TAAR family tree that is activated by isopentylamine, 2-phenylethylamine, p-tyramine, and agmatine significantly increased intracellular cAMP and resulted in increased insulin secretion from INS-1 cells and primary mouse islets under normal conditions. Crucially however, this enhancement was not evident when the receptor family was downregulated by glucolipotoxic conditions. This data indicates that a subset of TAARs are regulators of insulin secretion in pancreatic β-cells, and that their downregulation contributes to glucolipotoxic inhibition of insulin secretion. As such they may be potential targets for treatment of type 2 diabetes.

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN-γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN-γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN-γ-inducible MHC genes. Early responses to IFN-γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription.

Background BORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein. Results We show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes. Conclusion We have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

Background The discovery of novel anti-viral restriction factors illuminates unknown aspects of innate sensing and immunity. We identified RNA-associated Early-stage Anti-viral Factor (REAF) using a whole genome siRNA screen for restriction factors to human immunodeficiency virus (HIV) that act in the early phase of viral replication. Results We observed more than 50 fold rescue of HIV-1 infection, using a focus forming unit (FFU) assay, following knockdown of REAF by specific siRNA. Quantitative PCR was used to show that REAF knockdown results in an increase of early and late reverse transcripts which impacts the level of integration. REAF thus appears to act at an early stage of the viral life cycle during reverse transcription. Conversely when REAF is over-expressed in target cells less infected cells are detectable and fewer reverse transcripts are produced. Human REAF can also inhibit HIV-2 and simian immunodeficiency virus (SIV) infection. REAF associates with viral nucleic acids and may act to prevent reverse transcription. Conclusions This report firmly places REAF alongside APOBECs and SAMHD1 as a potent inhibitor of HIV replication acting early in the replication cycle, just after cell entry. We propose that REAF is part of an anti-viral surveillance system destroying incoming retroviruses. This novel mechanism could apply to invasion of cells by any intracellular pathogen.

BORIS (CTCFL) is the paralog of CTCF (CCCTC-binding factor; NM_006565), a ubiquitously expressed DNA-binding protein with diverse roles in gene expression and chromatin organisation. BORIS and CTCF have virtually identical zinc finger domains, yet display major differences in their respective C- and N-terminal regions. Unlike CTCF, BORIS expression has been reported only in the testis and certain malignancies, leading to its classification as a "cancer-testis" antigen. However, the expression pattern of BORIS is both a significant and unresolved question in the field of DNA binding proteins. Here, we identify BORIS in the cytoplasm and nucleus of a wide range of normal and cancer cells. We compare the localization of CTCF and BORIS in the nucleus and demonstrate enrichment of BORIS within the nucleolus, inside the nucleolin core structure and adjacent to fibrillarin in the dense fibrillar component. In contrast, CTCF is not enriched in the nucleolus. Live imaging of cells transiently transfected with GFP tagged BORIS confirmed the nucleolar accumulation of BORIS. While BORIS transcript levels are low compared to CTCF, its protein levels are readily detectable. These findings show that BORIS expression is more widespread than previously believed, and suggest a role for BORIS in nucleolar function.

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.