Search

Highlights • Virus manipulation of host processes can transform cells and trigger oncogenesis. • Viruses have evolved a variety of mechanism to hijack the host ubiquitin system. • Tumor viruses redirect ubiquitination to control the cellular DNA damage responses. • Viral-targeting of host cell DDR proteins can contribute to genome instability. • Viruses may exploit degradative or non-degradative outcomes within the DDR. Viruses exploit cellular ubiquitination machinery to shape the host proteome and promote productive infection. Among the cellular processes influenced by viral manipulation of ubiquitination is the DNA damage response (DDR), a network of cellular signaling pathways that sense and respond to genomic damage. This host–pathogen interaction is particularly important during virus replication and transformation by DNA tumor viruses. Manipulating DDR pathways can promote virus replication but also impacts host genomic instability, potentially leading to cellular transformation and tumor formation. We review ways in which viruses are known to hijack the cellular ubiquitin system to reshape host DDR pathways. [ABSTRACT FROM AUTHOR]

Summary Telomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1 and loss of the telomere repeat DNA signal. The HSV-1-encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. Small hairpin RNA (shRNA) depletion of TPP1 increases viral replication, indicating that TPP1 inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins, and ICP8-null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated prereplication foci and to promote viral genomic replication. [ABSTRACT FROM AUTHOR]

Abstract: During infection, viruses cause global disruption to nuclear architecture in their attempt to take over the cell. In turn, the host responds with various defenses, which include chromatin-mediated silencing of the viral genome and activation of DNA damage signaling pathways. Dynamic exchanges at chromatin, and specific post-translational modifications on histones have recently emerged as master controllers of DNA damage signaling and repair. Studying viral control of chromatin modifications is identifying histones as important players in the battle between host and virus for control of cell cycle and gene expression. These studies are revealing new complexities of the virus–host interaction, uncovering the potential of chromatin as an anti-viral defense mechanism, and also providing unique insights into the role of chromatin in DNA repair. [Copyright &y& Elsevier]

Abstract: Recognition and repair of DNA damage is critical for maintaining genomic integrity and suppressing tumorigenesis. In eukaryotic cells, the sensing and repair of DNA damage are coordinated with cell cycle progression and checkpoints, in order to prevent the propagation of damaged DNA. The carefully maintained cellular response to DNA damage is challenged by viruses, which produce a large amount of exogenous DNA during infection. Viruses also express proteins that perturb cellular DNA repair and cell cycle pathways, promoting tumorigenesis in their quest for cellular domination. This review presents an overview of strategies employed by viruses to manipulate DNA damage responses and cell cycle checkpoints as they commandeer the cell to maximize their own viral replication. Studies of viruses have identified key cellular regulators and revealed insights into molecular mechanisms governing DNA repair, cell cycle checkpoints, and transformation. [Copyright &y& Elsevier]

: BackgroundAngiopoietin-1 gene expression in human pulmonary hypertensive lungs is directly proportional to increasing pulmonary vascular resistance. We hypothesized that targeted overexpresssion of angiopoietin-1 in the lung would cause persistent pulmonary hypertension in an animal model.: MethodsWe injected 2 × 1010 genomic particles of adeno-associated virus-angiopoietin-1 (AAV-Ang-1) into the right ventricular outflow tract of 30 Fischer rats while using adeno-associated virus-lacZ (AAV-lacZ) injected rats and carrier-injected rats as our control groups. All animals underwent survival surgery and were sacrificed at serial timepoints postgene delivery. At each timepoint, pulmonary artery pressures were measured and pulmonary angiography using the Microfil polymer perfusion technique was performed. The lungs were harvested for pathologic analysis, mRNA analysis, Western blot assays, and in situ RNA hybridization to localize gene expression.: ResultsPulmonary artery pressures of AAV-Ang-1 injected rats were significantly increased compared with the control groups (p < 0.01) at all timepoints. Pathologic analysis of AAV-Ang-1 lung specimens demonstrated increased smooth muscle cell proliferation within the medial layer of arterioles with obliteration of small vessels similar to that seen in human pulmonary hypertension. Angiograms of AAV-Ang-1 injected lungs showed blunting of small peripheral arterioles consistent with advanced pulmonary hypertension. In situ RNA hybridization localized angiopoietin-1 expression to the vascular wall of small-caliber pulmonary vessels. Protein and mRNA assays confirmed persistent angiopoietin-1 expression in the lung for up to 60 days postgene delivery.: ConclusionsOverexpression of angiopoietin-1 using an adeno-associated virus vector causes pulmonary hypertension in rats. These data provide a novel physiologic animal model for pulmonary hypertension. [Copyright &y& Elsevier]

The APOBEC3 family of cytosine deaminases are part of the innate immune response to viral infection, but also have the capacity to damage cellular DNA. Detection of mutational signatures consistent with APOBEC3 activity, together with elevated APOBEC3 expression in cancer cells, has raised the possibility that these enzymes contribute to oncogenesis. Genome deamination by APOBEC3 enzymes also elicits DNA damage response signaling and presents therapeutic vulnerabilities for cancer cells. Here, we discuss implications of APOBEC3 activity in cancer and the potential to exploit their mutagenic activity for targeted cancer therapies. [ABSTRACT FROM AUTHOR]

Cellular SAMHD1 inhibits replication of many viruses by limiting intracellular deoxynucleoside triphosphate (dNTP) pools. We investigate the influence of SAMHD1 on human cytomegalovirus (HCMV). During HCMV infection, we observe SAMHD1 induction, accompanied by phosphorylation via viral kinase UL97. SAMHD1 depletion increases HCMV replication in permissive fibroblasts and conditionally permissive myeloid cells. We show this is due to enhanced gene expression from the major immediate-early (MIE) promoter and is independent of dNTP levels. SAMHD1 suppresses innate immune responses by inhibiting nuclear factor κB (NF-κB) activation. We show that SAMHD1 regulates the HCMV MIE promoter through NF-κB activation. Chromatin immunoprecipitation reveals increased RELA and RNA polymerase II on the HCMV MIE promoter in the absence of SAMHD1. Our studies reveal a mechanism of HCMV virus restriction by SAMHD1 and show how SAMHD1 deficiency activates an innate immune pathway that paradoxically results in increased viral replication through transcriptional activation of the HCMV MIE gene promoter. • HCMV infection induces SAMHD1 expression and phosphorylation • SAMHD1 restricts HCMV gene expression before virus replication • SAMHD1 deficiency limits HCMV entry into the quiescent stage of infection • HCMV restriction by SAMHD1 is mediated by limiting NF-κB activation SAMHD1 has been identified as a cellular antiviral restriction factor. Kim et al. report that HCMV is restricted by SAMHD1 through inhibition of viral gene expression. They show that depletion of SAMHD1 increases activation of the NF-κB immune pathway, which paradoxically increases gene expression from the immediate-early viral promoter. [ABSTRACT FROM AUTHOR]

The author discusses the goal of the American Society of Gene and Cell Therapy (ASGCT) to foster future leadership through creation and implementation of a comprehensive mentoring and support program for students, postdoctoral fellows and other new investigators. She mentions the role of mentoring in helping guide early-stage scientists toward opportunities that benefit their research. She also urges mentors to participate with the society develop therapies that will alleviate human disease.

During infection, viruses attempt to hijack the cell while the host responds with various defense systems. Traditional defenses include the interferon response and apoptosis, but recent work suggests that this antiviral arsenal also includes the cellular DNA damage response machinery. The observation of interactions between viruses and cellular DNA repair proteins has not only uncovered new complexities of the virus–host interaction but is also reinforcing the view that viruses can reveal key regulators of cellular pathways through the proteins they target. [Copyright &y& Elsevier]

Highlights • The protein-linked adenovirus genome was used to study repair of 5′ protein-linked DNA double strand breaks (DSBs) in vivo. • Adenovirus terminal protein (TP) is endonucleolytically removed from the genome of the adenovirus dl 1004 mutant. • Mre11 acts as the nuclease to release TP, but is dependent upon the presence of CtIP. • Similar to Top2 and Spo11, liberated adenovirus TP remains attached to 16–25mer oligonucleotides. Abstract The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5′ termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5′ terminus of its dsDNA genome, for studying the repair of 5′ protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo , suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl 1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5′ termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5′ termini of DSBs. [ABSTRACT FROM AUTHOR]

Abstract: Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells. [Copyright &y& Elsevier]

Recombinant cross-packaging of adeno-associated virus (AAV) genome of one serotype into other AAV serotypes has the potential to optimize tissue-specific gene transduction and expression in the heart. To evaluate the role of AAV1 to 5 virion shells on AAV2 transgene transduction, we constructed hybrid vectors in which each serotype capsid coding domain was cloned into a common vector backbone containing AAV2 replication genes. Constructs were tested for expression in: (1) adult murine heart in vivo using direct injection of virus, (2) neonatal and adult murine ventricular cardiomyocytes in vitro, and (3) adult human ventricular cardiomyocytes in vitro, using green fluorescent protein (GFP) as the measurable transgene. Serotype 1 virus demonstrated the highest transduction efficiency in adult murine cardiomyocytes both in vitro and in vivo, while serotype 2 virus had the greater transduction efficiency in neonatal cardiomyocytes in vitro. Prolonged in vivo myocardial GFP expression was observed for up to 12 months using serotype 1 and 2 vectors only. In human cardiomyocytes, serotype 1 vector was superior in transduction efficiency, followed by types 2, 5, 4, and 3. These data establish a hierarchy for efficient serotype-specific vector transduction in myocardial tissue. AAV1 serotype packaging results in more efficient transduction of genes in the murine and human adult heart, compared to other AAV serotypes. Our results suggest that adult human cardiac gene therapy may be enhanced by the use of serotype 1-specific AAV vectors. [Copyright &y& Elsevier]

An abstract of the article "Optimizing Custom Zinc-Finger Nucleases for Use in Human Cells," by Stephen Alwin, Maja B. Gere, Eva Guhl, Karin Effertz, Carlos F. Barbas III, David J. Segal, Matthew D. Weitzman, and Toni Cathomen is presented.