SUMO pathway dependent recruitment of cellular repressors to herpes simplex virus type 1 genomes.

Components of promyelocytic leukaemia (PML) nuclear bodies (ND10) are recruited to sites associated with herpes simplex virus type 1 (HSV-1) genomes soon after they enter the nucleus. This cellular response is linked to intrinsic antiviral resistance and is counteracted by viral regulatory protein ICP0. We report that the SUMO interaction motifs of PML, Sp100 and hDaxx are required for recruitment of these repressive proteins to HSV-1 induced foci, which also contain SUMO conjugates and PIAS2ß, a SUMO E3 ligase. SUMO modification of PML and elements of its tripartite motif (TRIM) are also required for recruitment in cells lacking endogenous PML. Mutants of PML isoform I and hDaxx that are not recruited to virus induced foci are unable to reproduce the repression of ICP0 null mutant HSV-1 infection mediated by their wild type counterparts. We conclude that recruitment of ND10 components to sites associated with HSV-1 genomes reflects a cellular defence against invading pathogen DNA that is regulated through the SUMO modification pathway.

Regulation of ICP0-null mutant herpes simplex virus type 1 infection by ND10 components ATRX and hDaxx.

Herpes simplex virus type 1 (HSV-1) immediate-early gene product ICP0 activates lytic infection and relieves cell-mediated repression of viral gene expression. This repression is conferred by preexisting cellular proteins and is commonly referred to as intrinsic antiviral resistance or intrinsic defense. PML and Sp100, two core components of nuclear substructures known as ND10 or PML nuclear bodies, contribute to intrinsic resistance, but it is clear that other proteins must also be involved. We have tested the hypothesis that additional ND10 factors, particularly those that are involved in chromatin remodeling, may have roles in intrinsic resistance against HSV-1 infection. The two ND10 component proteins investigated in this report are ATRX and hDaxx, which are known to interact with each other and comprise components of a repressive chromatin-remodeling complex. We generated stable cell lines in which endogenous ATRX or hDaxx expression is severely suppressed by RNA interference. We found increases in both gene expression and plaque formation induced by ICP0-null mutant HSV-1 in both ATRX- and hDaxx-depleted cells. Reconstitution of wild-type hDaxx expression reversed the effects of hDaxx depletion, but reconstitution with a mutant form of hDaxx unable to interact with ATRX did not. Our results suggest that ATRX and hDaxx act as a complex that contributes to intrinsic antiviral resistance to HSV-1 infection, which is counteracted by ICP0.

Molecular characterization of HEK293 cells as emerging versatile cell factories.

HEK293 cell lines are used for the production of recombinant proteins, virus-like particles and viral vectors. Recent work has generated molecular (systems level) characterisation of HEK293 variants that has enabled re-engineering of the cells towards enhanced use for manufacture-scale production of recombinant biopharmaceuticals (assessment of 'safe harbours' for gene insertion, engineering of new variants for stable, amplifiable expression). In parallel, there have been notable advances in the bioprocessing conditions (suspension adaptation, development of defined serum-free media) that offer the potential for large-scale manufacture, a feature especially important in the drive to produce viral vectors at large-scale and at commercially viable costs for gene therapy. The combination of cell-based and bioprocess-based modification of existing HEK293 cell processes, frequently informed by understandings transferred from developments with Chinese hamster ovary cell lines, seems destined to place the HEK293 cell systems firmly as a critical platform for production of future biologically based therapeutics.

Human cytomegalovirus protein pp71 displaces the chromatin-associated factor ATRX from nuclear domain 10 at early stages of infection.

The human cytomegalovirus (HCMV) tegument protein pp71, encoded by gene UL82, stimulates viral immediate-early (IE) transcription. pp71 interacts with the cellular protein hDaxx at nuclear domain 10 (ND10) sites, resulting in the reversal of hDaxx-mediated repression of viral transcription. We demonstrate that pp71 displaces an hDaxx-binding protein, ATRX, from ND10 prior to any detectable effects on hDaxx itself and that this event contributes to the role of pp71 in alleviating repression. Introduction of pp71 into cells by transfection, infection with a pp71-expressing herpes simplex virus type 1 vector, or by generation of transformed cell lines promoted the rapid relocation of ATRX from ND10 to the nucleoplasm without alteration of hDaxx levels or localization. A pp71 mutant protein unable to interact with hDaxx did not affect the intranuclear distribution of ATRX. Infection with HCMV at a high multiplicity of infection resulted in rapid displacement of ATRX from ND10, the effect being observed maximally by 2 h after adsorption, whereas infection with the UL82-null HCMV mutant ADsubUL82 did not affect ATRX localization even at 7 h postinfection. Cell lines depleted of ATRX by transduction with shRNA-expressing lentiviruses supported increased IE gene expression and virus replication after infection with ADsubUL82, demonstrating that ATRX has a role in repressing IE transcription. The results show that ATRX, in addition to hDaxx, is a component of cellular intrinsic defenses that limit HCMV IE transcription and that displacement of ATRX from ND10 by pp71 is important for the efficient initiation of viral gene expression.

C9orf72 expansion disrupts ATM-mediated chromosomal break repair.

Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.

AAV9-mediated central nervous system-targeted gene delivery via cisterna magna route in mice.

Current barriers to the use of adeno-associated virus serotype 9 (AAV9) in clinical trials for treating neurological disorders are its high expression in many off-target tissues such as liver and heart, and lack of cell specificity within the central nervous system (CNS) when using ubiquitous promoters such as human cytomegalovirus (CMV) or chicken-ß-actin hybrid (CAG). To enhance targeting the transgene expression in CNS cells, self-complementary (sc) AAV9 vectors, scAAV9-GFP vectors carrying neuronal Hb9 and synapsin 1, and nonspecific CMV and CAG promoters were constructed. We demonstrate that synapsin 1 and Hb9 promoters exclusively targeted neurons in vitro, although their strengths were up to 10-fold lower than that of CMV. In vivo analyses of mouse tissue after scAAV9-GFP vector delivery via the cisterna magna revealed a significant advantage of synapsin 1 promoter over both Hb9 variants in targeting neurons throughout the brain, since Hb9 promoters were driving gene expression mainly within the motor-related areas of the brain stem. In summary, this study demonstrates that cisterna magna administration is a safe alternative to intracranial or intracerebroventricular vector delivery route using scAAV9, and introduces a novel utility of the Hb9 promoter for the targeted gene expression for both in vivo and in vitro applications.