The roles of ND10 proteins ATRX and hDaxx in the regulation of herpesvirus infection

Protection against viruses is provided by the host innate, adaptive and cellular intrinsic immunities. Cellular intrinsic immunity, or intrinsic defence, against herpesviruses is in part enacted by components that constitute nuclear substructures known as ND10. ND10 (also called PML nuclear bodies after its major organising component) are dynamic nuclear domains that contain various cellular proteins, including PML itself, Sp100, hDaxx and ATRX. In the early stages of infection, herpesvirus genomes and sites of immediate early transcription become associated with ND10 and their components. Representative herpesviruses such as an alphaherpesvirus HSV-1 and a betaherpesvirus HCMV encode for strong transcriptional activators, namely the Immediate Early protein ICP0 and the tegument protein pp71, respectively. These proteins are known to counteract cellular intrinsic defence mechanisms. In the context of HSV-1 and HCMV infections, the major ND10 component PML has been identified as an important constituent of cellular intrinsic defence. In addition, a number of research studies have demonstrated that Sp100 and hDaxx contribute to these processes during infection with HSV-1 and HCMV, respectively. The general hypothesis of the present study implies that cellular chromatin-associated factors within ND10 may act to repress viral gene expression. The two representative ND10 components present a particular interest for the current investigation based on the facts that: (i) hDaxx is a transcriptional co-repressor; (ii) ATRX is a chromatin-remodelling enzyme; and (iii) ATRX and hDaxx interact with each other to form a chromatin-remodelling complex with repressive properties. The purpose of the present study was therefore to investigate the roles of these two proteins in HSV-1 and HCMV infection. By using virus mutants incapable of efficient stimulation of Immediate Early gene expression (ICP0-null HSV-1 and pp71-null HCMV) it was possible to analyse the contribution of ATRX and hDaxx to the repression mechanism that occurs in the absence of these viral transactivators. An RNA interference approach was utilised for generating cell lines depleted of ATRX or hDaxx in order to assess their roles in viral infectivity. In addition, cell lines reconstituted with wt hDaxx, and ATRX-interaction or SUMO-interaction deficient hDaxx mutants were constructed in order to study the contribution of these functional elements to the role of hDaxx in the repression of ICP0-null mutant HSV-1. The key findings presented in the current study can be summarised as follows: (1) ATRX contributes to the intrinsic resistance against HCMV infection, and this mechanism is strongly counteracted by viral pp71; (2) A chromatin-remodelling complex formed between ATRX and hDaxx contributes to the efficient repression of ICP0-null HSV-1 genomes, thereby constituting a part of anti-HSV-1 intrinsic cellular defence. ICP0 counteracts this process, and the possible mechanisms of ICP0 action are proposed. These data provide the first evidence for the role of ATRX in viral infection and in addition demonstrate a role for hDaxx in the regulation of HSV-1 infection. The strong indication that ATRX and hDaxx act as a complex opens a possibility of chromatin-dependent repression of ICP0-null HSV-1 genomes. Whether ATRX and hDaxx contribute to the repression of pp71-null HCMV genomes as a complex is yet to be established. In summary, the conclusion of the studies presented in this thesis suggests regulatory roles of ND10-localised chromatin remodelling proteins ATRX and hDaxx in cellular anti-herpesvirus intrinsic resistance mechanisms

Enhancing rAAV production by HEK293 cells via metabolic profiling

Viral vector manufacturing is expensive and time-consuming. Demand for rAAV-based vectors has risen massively in the past decade and continues to rise thanks to urgent healthcare supply demand. The industry is, however, currently missing a cost-effective and robust manufacturing strategy. One of the major downsides of rAAV production is the high percentage of “empty” vector particles being produced and harvested. In addition to complicating downstream purification processes, this characteristic limits the efficiency of rAAV manufacture and presents uncertainties for scale-up. Efficiency of the manufacturing process is largely dependent on the productivity of the production cell line. Much emphasis has been put into understanding the effects of recombinant protein production on mammalian cell lines (e.g., CHO, HeLa, HEK293) but relatively little is known about the effects of viral vector production on cell biology and behaviour. Over the years, many clones have been derived, isolated and engineered from HEK293 to induce improvements in productivity and efficiency. However, the high cost of production and licensing, the expression of potentially undesired elements (e.g., T-antigen) and regulatory approval processes for next generation cell lines, hinders their use in clinical manufacturing. Increased understanding of HEK293 in relation to existing processes and process control offers realistic opportunity to enhance the efficiency of rAAV manufacturing. Our aim is to identify and understand the critical parameters that contribute to setting the productivity in HEK293 cells (in terms of final yield and abundance of full capsids), ranging from the metabolic requirements prior to and during viral vector production, to cell culture parameter optimisation to maintain the cells in an optimal state of health. We tested several commercially available media for rAAV9 production and selected the candidate that provided the best yield and quality of viral vector. With this medium as our baseline, we investigated the metabolism during a period of culture via extracellular metabolic profiling of control and rAAV producing cells. The analysis revealed the rapid use of several amino acids over the first 24 hr post-inoculation and the subsequent generation of metabolites indicative of metabolic profiles associated with cell growth. rAAV9 producing cells show lower rates of amino acid and glucose consumption than control cells but the profile of metabolism was not significantly changed as a result of transfection/production of rAAV9. These data were used to design medium supplements and the effect of supplement addition on cell proliferation, viability and rAAV production/quality was assessed. Specific combinations of amino acids generated an increased cell density (up to 9.3x106 cells/mL at 5 days post-inoculation compared to 4.4x106 cells/mL for cells in non-supplemented medium). This was associated with retention of improved viability in the presence of the supplement. In addition, the metabolic profiling we undertook indicated the build-up of potentially toxic/growth inhibitory metabolites during the period of stock cell preparations prior to setting up transfections. In various dilution experiments we were able to optimise the pre-treatment, cell density and dilution protocol to generate predictable and reproducible efficiencies of transfection, cell growth and rAAV production. Overall, our data contributes metabolic insights to process conditions that generate HEK293 cells of appropriate health and defined parameters to robust and enhanced production of rAAV, providing work schemes that are also appropriate to the manufacture of further types of viral vectors

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 PIAS2β, 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

C9orf72 Expansion Disrupts ATM-mediated Chromosomal Break Repair

A hexanucleotide repeat expansion represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which the expansion cause neurodegeneration are poorly understood. We report elevated levels of DNA/RNA hybrids (R-loops) and double-strand breaks (DSBs) in rodent neurons, human cells, and in C9orf72-ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signalling and accumulation of protein-linked DNA breaks. We further reveal that defective ATM-mediated DNA repair is a consequence of p62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signalling. Adeno-associated virus- mediated expression of C9orf72-related RNA and dipeptide repeats in the murine central nervous system causes elevated DSBs, ATM defects, and triggers neurodegeneration. These findings identify R-Loops, DSBs, and defective ATM-mediated repair as pathological consequences of C9orf72 expansions, and suggest that C9orf72-linked neurodegeneration is driven, at least in part, by genomic instability

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.

From PubMed via Jisc Publications RouterHistory: received 2021-03-05, revised 2021-04-29, accepted 2021-05-07Publication status: aheadofprintHEK293 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. [Abstract copyright: Copyright © 2021 Elsevier Ltd. All rights reserved.

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

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