Circularization of the HIV-1 RNA genome

Genomic RNA circularization has been proposed for several RNA viruses. In this study, we examined if the 5′ and 3′ ends of the 9-kb HIV-1 RNA genome can interact. In vitro assays demonstrated a specific interaction between transcripts encompassing the 5′ and 3′ terminal 1 kb, suggesting that the HIV-1 RNA genome can circularize. Truncation of the transcripts indicated that the 5′–3′ interaction is formed by 600–700 nt in the gag open reading frame and the terminal 123 nt of the genomic RNA. Detailed RNA structure probing indicates that sequences flanking the 3′ TAR hairpin interact with complementary sequences in the gag gene. Phylogenetic analysis indicates that all HIV-1 subtypes can form the 5′/3′ interaction despite considerable sequence divergence, suggesting an important role of RNA circularization in the HIV-1 replication cycle

DDX5 potentiates HIV-1 transcription as a co-factor of Tat

Funder: Cambridge Biomedical Research Centre (GB)Funder: Clinical Academic ReserveAbstract: Background: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein–protein interactions and RNA and ATP binding. Results: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. Conclusion: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1

DDX5 potentiates HIV-1 transcription as a co-factor of Tat

Funder: Cambridge Biomedical Research Centre (GB)Funder: Clinical Academic ReserveAbstract: Background: HIV-1 does not encode a helicase and hijacks those of the cell for efficient replication. We and others previously showed that the DEAD box helicase, DDX5, is an essential HIV dependency factor. DDX5 was recently shown to be associated with the 7SK snRNP. Cellular positive transcription elongation factor b (P-TEFb) is bound in an inactive form with HEXIM1/2 on 7SK snRNP. The Tat/P-TEFb complex is essential for efficient processivity of Pol II in HIV-1 transcription elongation and Tat competes with HEXIM1/2 for P-TEFb. We investigated the precise role of DDX5 in HIV replication using siRNA mediated knockdown and rescue with DDX5 mutants which prevent protein–protein interactions and RNA and ATP binding. Results: We demonstrate a critical role for DDX5 in the Tat/HEXIM1 interaction. DDX5 acts to potentiate Tat activity and can bind both Tat and HEXIM1 suggesting it may facilitate the dissociation of HEXIM1/2 from the 7SK-snRNP complex, enhancing Tat/P-TEFb availability. We show knockdown of DDX5 in a T cell line significantly reduces HIV-1 infectivity and viral protein production. This activity is unique to DDX5 and cannot be substituted by its close paralog DDX17. Overexpression of DDX5 stimulates the Tat/LTR promoter but suppresses other cellular and viral promoters. Individual mutations of conserved ATP binding, RNA binding, helicase related or protein binding motifs within DDX5 show that the N terminal RNA binding motifs, the Walker B and the glycine doublet motifs are essential for this function. The Walker A and RNA binding motifs situated on the transactivation domain are however dispensable. Conclusion: DDX5 is an essential cellular factor for efficient HIV transcription elongation. It interacts with Tat and may potentiate the availability of P-TEFb through sequestering HEXIM1

Umberto Eco, Narratives of Conspiracy and the Anti-Semite Among Us

http://www.quest-cdecjournal.it/discussion.php?id=3

Exome sequencing reveals mutated SLC19A3 in patients with an early-infantile, lethal encephalopathy

To accomplish a diagnosis in patients with a rare unclassified disorder is difficult. In this study, we used magnetic resonance imaging pattern recognition analysis to identify patients with the same novel heritable disorder. Whole-exome sequencing was performed to discover the mutated gene. We identified seven patients sharing a previously undescribed magnetic resonance imaging pattern, characterized by initial swelling with T2 hyperintensity of the basal nuclei, thalami, cerebral white matter and cortex, pons and midbrain, followed by rarefaction or cystic degeneration of the white matter and, eventually, by progressive cerebral, cerebellar and brainstem atrophy. All patients developed a severe encephalopathy with rapid deterioration of neurological functions a few weeks after birth, followed by respiratory failure and death. Lactate was elevated in body fluids and on magnetic resonance spectroscopy in most patients. Whole-exome sequencing in a single patient revealed two predicted pathogenic, heterozygous missense mutations in the SLC19A3 gene, encoding the second thiamine transporter. Additional predicted pathogenic mutations and deletions were detected by Sanger sequencing in all six other patients. Pathology of brain tissue of two patients demonstrated severe cerebral atrophy and microscopic brain lesions similar to Leigh's syndrome. Although the localization of SLC19A3 expression in brain was similar in the two investigated patients compared to age-matched control subjects, the intensity of the immunoreactivity was increased. Previously published patients with SLC19A3 mutations have a milder clinical phenotype, no laboratory evidence of mitochondrial dysfunction and more limited lesions on magnetic resonance imaging. In some, cerebral atrophy has been reported. The identification of this new, severe, lethal phenotype characterized by subtotal brain degeneration broadens the phenotypic spectrum of SLC19A3 mutations. Recognition of the associated magnetic resonance imaging pattern allows a fast diagnosis in affected infant

Case Series and DARS2 Variant Analysis in Early Severe Forms With Unexpected Presentations

Objective: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is regarded a relatively mild leukodystrophy, diagnosed by characteristic long tract abnormalities on MRI and biallelic variants in DARS2, encoding mitochondrial aspartyl-tRNA synthetase (mtAspRS). DARS2 variants in LBSL are almost invariably compound heterozygous; in 95% of cases, 1 is a leaky splice site variant in intron 2. A few severely affected patients, still fulfilling the MRI criteria, have been described. We noticed highly unusual MRI presentations in 15 cases diagnosed by WES. We examined these cases to determine whether they represent consistent novel LBSL phenotypes. Methods: We reviewed clinical features, MRI abnormalities, and gene variants and investigated the variants' impact on mtAspRS structure and mitochondrial function. Results: We found 2 MRI phenotypes: early severe cerebral hypoplasia/atrophy (9 patients, group 1) and white matter abnormalities without long tract involvement (6 patients, group 2). With antenatal onset, microcephaly, and arrested development, group 1 patients were most severely affected. DARS2 variants were severer than for classic LBSL and severer for group 1 than group 2. All missense variants hit mtAspRS regions involved in tRNAAsp binding, aspartyl-adenosine-5'-monophosphate binding, and/or homodimerization. Missense variants expressed in the yeast DARS2 ortholog showed severely affected mitochondrial function. Conclusions: DARS2 variants are associated with highly heterogeneous phenotypes. New MRI presentations are profound cerebral hypoplasia/atrophy and white matter abnormalities without long tract involvement. Our findings have implications for diagnosis and understanding disease mechanisms, pointing at dominant neuronal/axonal involvement in severe cases. In line with this conclusion, activation of biallelic DARS2 null alleles in conditional transgenic mice leads to massive neuronal apoptosis

Ribosomal scanning on the 5′-untranslated region of the human immunodeficiency virus RNA genome

Translation initiation on most eukaryotic mRNAs occurs via a cap-dependent scanning mechanism and its efficiency is modulated by their 5′-untranslated regions (5′-UTR). The human immunodeficiency virus type 1 (HIV-1) 5′-UTR contains a stable TAR hairpin directly at its 5′-end, which possibly masks the cap structure. In addition, the 5′-UTR is relatively long and contains several stable RNA structures that are essential for viral replication. These characteristics may interfere with ribosomal scanning and suggest that translation is initiated via internal entry of ribosomes. Literature on the HIV-1 5′-UTR-driven translation initiation mechanism is controversial. Both scanning and internal initiation have been shown to occur in various experimental systems. To gain further insight in the translation initiation process, we determined which part of the 5′-UTR is scanned. To do so, we introduced upstream AUGs at various positions across the 5′-UTR and determined the effect on expression of a downstream reporter gene that was placed under control of the gag start codon. This strategy allowed us to determine the window of ribosomal scanning on the HIV-1 5′-UTR

Aquaporin-4 and GPRC5B: old and new players in controlling brain oedema

Brain oedema is a life-threatening complication of various neurological conditions. Understanding molecular mechanisms of brain volume regulation is critical for therapy development. Unique insight comes from monogenic diseases characterized by chronic brain oedema, of which megalencephalic leukoencephalopathy with subcortical cysts (MLC) is the prototype. Variants in MLC1 or GLIALCAM, encoding proteins involved in astrocyte volume regulation, are the main causes of MLC. In some patients, the genetic cause remains unknown. We performed genetic studies to identify novel gene variants in MLC patients, diagnosed by clinical and MRI features, without MLC1 or GLIALCAM variants. We determined subcellular localization of the related novel proteins in cells and in human brain tissue. We investigated functional consequences of the newly identified variants on volume regulation pathways using cell volume measurements, biochemical analysis and electrophysiology. We identified a novel homozygous variant in AQP4, encoding the water channel aquaporin-4, in two siblings, and two de novo heterozygous variants in GPRC5B, encoding the orphan G protein-coupled receptor GPRC5B, in three unrelated patients. The AQP4 variant disrupts membrane localization and thereby channel function. GPRC5B, like MLC1, GlialCAM and aquaporin-4, is expressed in astrocyte endfeet in human brain. Cell volume regulation is disrupted in GPRC5B patient-derived lymphoblasts. GPRC5B functionally interacts with ion channels involved in astrocyte volume regulation. In conclusion, we identify aquaporin-4 and GPRC5B as old and new players in genetic brain oedema. Our findings shed light on the protein complex involved in astrocyte volume regulation and identify GPRC5B as novel potentially druggable target for treating brain oedema

RNA Structure Modulates Splicing Efficiency at the Human Immunodeficiency Virus Type 1 Major Splice Donor▿

The untranslated leader of the human immunodeficiency virus type 1 (HIV-1) RNA genome encodes essential sequence and structural motifs that control various replication steps. The 5′ splice site or splice donor (SD) is embedded in a semistable hairpin, but the function of this structure is unknown. We stabilized this SD hairpin by creating an additional base pair and demonstrated a severe HIV-1 replication defect. A splicing defect was apparent in RNA analyses of virus-infected cells and cells transfected with appropriate reporter constructs. We selected multiple virus revertants in search for interesting second-site escape pathways. Most revertants acquired an additional mutation that modulated the stability of the mutant SD hairpin. One revertant acquired a single nucleotide change in the upstream DIS hairpin. We demonstrate that a novel SD site is created by this upstream mutation, which obviously reduces the number of leader nucleotides that are included in spliced HIV-1 transcripts. These results suggest a novel role of RNA structure in the regulation of HIV-1 splicing