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Abstract: Viruses often induce signaling through the same cellular cascades that are activated by damage to the cellular genome. Signaling triggered by viral proteins or exogenous DNA delivered by viruses can be beneficial or detrimental to viral infection. Viruses have therefore evolved to dissect the cellular DNA damage response pathway during infection, often marking key cellular regulators with ubiquitin to induce their degradation or change their function. Signaling controlled by ubiquitin or ubiquitin-like proteins has recently emerged as key regulator of the cellular DNA damage response. Situated at the interface between DNA damage signaling and the ubiquitin system, viruses can reveal key convergence points in this important cellular pathway. In this review, we examine how viruses harness the diversity of the cellular ubiquitin system to modulate the DNA damage signaling pathway. We discuss the implications of viral infiltration of this pathway for both the transcriptional program of the virus and for the cellular response to DNA damage. [Copyright &y& Elsevier]

Human apolipoprotein-B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins constitute a family of cytidine deaminases that mediate restriction of retroviruses, endogenous retro-elements and DNA viruses. It is well established that these enzymes are potent mutators of viral DNA, but it is unclear whether their editing activity is a threat to the integrity of the cellular genome. We show that expression of APOBEC3A can lead to induction of DNA breaks and activation of damage responses in a deaminase-dependent manner. Consistent with these observations, APOBEC3A expression induces cell-cycle arrest. These results indicate that cellular DNA is vulnerable to APOBEC3 activity and deregulated expression of APOBEC3A could threaten genomic integrity. [ABSTRACT FROM AUTHOR]

Abstract: Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein–DNA complexes known as telomeres. The Mre11–Rad50–Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end-joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here, we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction. [ABSTRACT FROM AUTHOR]

The cellular surveillance network for sensing and repairing damaged DNA prevents an array of human diseases, and when compromised it can lead to genomic instability and cancer. The carefully maintained cellular response to DNA damage is challenged during viral infection, when foreign DNA is introduced into the cell. The battle between virus and host generates a genomic conflict. The host attempts to limit viral infection and protect its genome, while the virus deploys tactics to eliminate, evade, or exploit aspects of the cellular defense. Studying this conflict has revealed that the cellular DNA damage response machinery comprises part of the intrinsic cellular defense against viral infection. In this review we examine recent advances in this emerging field. We identify common themes used by viruses in their attempts to commandeer or circumvent the host cell's DNA repair machinery, and highlight potential outcomes of the conflict for both virus and host. [ABSTRACT FROM AUTHOR]

The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes. [ABSTRACT FROM AUTHOR]

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection. [ABSTRACT FROM AUTHOR]

The Schlafen gene family encodes for proteins involved in various biological tasks, including cell proliferation, differentiation, and T cell development. Schlafens were initially discovered in mice, and have been studied in the context of cancer biology, as well as their role in protecting cells during viral infection. This protein family provides antiviral barriers via direct and indirect effects on virus infection. Schlafens can inhibit the replication of viruses with both RNA and DNA genomes. In this review, we summarize the cellular functions and the emerging relationship between Schlafens and innate immunity. We also discuss the functions and distinctions of this emerging family of proteins as host restriction factors against viral infection. Further research into Schlafen protein function will provide insight into their mechanisms that contribute to intrinsic and innate host immunity. [ABSTRACT FROM AUTHOR]

DNA viruses promote cell cycle progression, stimulate unscheduled DNA synthesis, and present the cell with an extraordinary amount of exogenous DNA. These insults elicit vigorous responses mediated by cellular factors that govern cellular homeostasis. To ensure productive infection, adenovirus has developed means to inactivate these intracellular antiviral responses. Among the challenges to the host cell is the viral DNA genome, which is viewed as DNA damage and elicits a cellular response to inhibit replication. Adenovirus therefore encodes proteins that dismantle the cellular DNA damage machinery. Studying virus–host interactions has yielded insights into the molecular functioning of fundamental cellular mechanisms. In addition, it has suggested ways that viral cytotoxicity can be exploited to offer a selective means of restricted growth in tumor cells as a therapy against cancer. In this review, we discuss aspects of the intracellular response that are unique to adenovirus infection and how adenoviral proteins produced from the early region E4 act to neutralize antiviral defenses, with a particular focus on DNA damage signaling.Oncogene (2005) 24, 7686–7696. doi:10.1038/sj.onc.1209063 [ABSTRACT FROM AUTHOR]

Broadly defined, the concept of gene therapy involves the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery. Their performance and pathogenicity has been evaluated in animal models, and encouraging results form the basis for clinical trials to treat genetic disorders and acquired diseases. Despite some initial success in these trials, vector development remains a seminal concern for improved gene therapy technologies. [ABSTRACT FROM AUTHOR]

We report that herpes simplex virus 1 (HSV-1) infection can activate and exploit a cellular DNA damage response that aids viral replication in nonneuronal cells Early in HSV-1 infection, several members of the cellular DNA damage-sensing machinery are activated and accumulate at sites of viral DNA replication. When this cellular response is abrogated, formation of HSV-1 replication centers is retarded, and viral production is compromised. In neurons, HSV-1 replication centers fail to mature, and the DNA damage response is not initiated. These data suggest that the failure of neurons to mount a DNA damage response to HSV-1 may contribute to the establishment of latency. [ABSTRACT FROM AUTHOR]

Presents a study which examined the significance of adenovirus oncoproteins to Mre-11, Rad50 and NBS1 DNA repair complex. Examination of cellular proteins required for concatemerization; Assessment of the effect of adenovirus infection on the localization of the DNA complex; Mechanisms required in preventing concatemer formation.

Describes a design of a genetic screen to identify cellular Rep recognition sequence (RRS)-binding proteins. Presence of RRS proteins in the cellular adeno-associated virus type 1 (AAV1) integration locus; Reactivity of human zinc finger protein with RRS motifs; Contribution of ZF5 to AAV2 repression.

Adenovirus is a nuclear replicating DNA virus reliant on host RNA processing machinery. Processing and metabolism of cellular RNAs can be regulated by METTL3, which catalyzes the addition of N6-methyladenosine (m6A) to mRNAs. While m6A-modified adenoviral RNAs have been previously detected, the location and function of this mark within the infectious cycle is unknown. Since the complex adenovirus transcriptome includes overlapping spliced units that would impede accurate m6A mapping using short-read sequencing, here we profile m6A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m6A detection. Although both early and late viral transcripts contain m6A, depletion of m6A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m6A discovery within individual transcripts at nucleotide resolution, and highlight the role of m6A in regulating splicing of a viral pathogen. Adenovirus transcripts contain N6-methyladenosine (m6A) modification. Here the authors profile m6A modification sites on adenovirus mRNAs using Illumina meRIP-Seq and nanopore direct RNA sequencing, and showcase a role for m6A in splicing of viral late mRNAs. [ABSTRACT FROM AUTHOR]

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A. Synopsis: APOBEC3A is a prominent source of mutagenesis across many cancer types. This study defines a novel role of the SMC5/6 complex in maintaining genomic stability and viability of cancer cells in which APOBEC3A is active, revealing a potential therapeutic vulnerability. In the absence of functional SMC5/6, APOBEC3A activity results in genotoxic cell death SMC5/6 dysfunction augments APOBEC3A-mediated DNA damage during replication PrimPol generates single-strand DNA gaps upon APOBEC3A activity at replication forks SMC5/6 may play a critical role in maintaining genome stability during APOBEC3A activity by stabilization of replication forks containing single-strand DNA gaps Maintaining genome stability in the presence of active APOBEC3A involves SMC5/6-dependent stabilization of forks with single-strand gaps, suggesting a potential vulnerability of cancer cells. [ABSTRACT FROM AUTHOR]

Biomolecular condensates (BMCs) play important roles in diverse biological processes. Many viruses form BMCs which have been implicated in various functions critical for the productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral BMCs that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquidlike properties. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and the production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral BMCs to limit viral multiplication. [ABSTRACT FROM AUTHOR]

Genomic rearrangements contribute to the enhancement of genetic diversity in populations. However, non-canonical rearrangements (NCRs) such as deletions, insertions, and inversions have the potential to trigger genomic instability. In the case of DNA viruses, NCRs can lead to generation of defective viral genomes (DVGs). To study NCRs in herpes simplex virus type 1 (HSV-1) genomes, we enriched DVGs formation by undiluted serial passaging on various cell types. We found that viral passaging on cell type that enables more viral genomes to initiate replication induces higher amplitude and frequency of cyclic patterns associated with DVGs formation. Despite differences in the rates of DVG accumulation, cell lines displayed comparable quantities of distinct NCRs, indicating that fluctuations caused by DVGs may impose bottlenecks on population genetic diversity. These findings propose additional roles for DVGs in modulating viral genetic diversity. Each cell type exhibited a unique population of NCRs, suggesting that NCRs accumulate in a cell type-specific manner. Interestingly, we identified a higher prevalence of short homologies and short reverse complementary in the parental sequences of NCR junction sites across all cell types. These shared sequence characteristics were also observed in NCRs identified in sequences obtained from clinical samples. The fundamental properties of HSV-1 NCR formation uncovered in this study may have broader implications for other DNA viruses. [ABSTRACT FROM AUTHOR]

The interest in MS-based analysis of modified nucleic acids is increasing due to the application of nucleic acids in therapeutics. However, there are few available integrated platforms for characterizing nucleic acid modifications. Herein, we report a general mass spectrometry-based SWATH platform to identify and quantify both RNA and DNA modifications, which we call SWATH analysis of modified nucleic acids (SWAMNA). SWAMNA incorporates the search engine, NuMo finder, enabling the analysis of modifications in native and permethylated form. SWAMNA will aid discoveries that provide new insights into nucleic acid modifications. [ABSTRACT FROM AUTHOR]

Viruses regulate cellular processes to facilitate viral replication. Manipulation of nuclear proteins and pathways by nuclear replicating viruses often causes cellular genome instability that contributes to transformation. The cellular DNA damage response (DDR) safeguards the host to maintain genome integrity, but DNA tumour viruses can manipulate the DDR to promote viral propagation. In this review, we describe the interactions of DNA tumour viruses with the phosphatidylinositol 3-kinase-like protein kinase (PIKK) pathways, which are central regulatory arms of the DDR.We review how signalling through the ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), and DNA-dependent protein kinases (DNA-PK) influences viral life cycles, and how their manipulation by viral proteins may contribute to tumour formation. [ABSTRACT FROM AUTHOR]

Herpes simplex virus (HSV) infection of the neonatal brain causes severe encephalitis and permanent neurologic deficits. However, infants infected with HSV at the time of birth follow varied clinical courses, with approximately half of infants experiencing only external infection of the skin rather than invasive neurologic disease. Understanding the cause of these divergent outcomes is essential to developing neuroprotective strategies. To directly assess the contribution of viral variation to neurovirulence, independent of human host factors, we evaluated clinical HSV isolates from neonates with different neurologic outcomes in neurologically relevant in vitro and in vivo models. We found that isolates taken from neonates with encephalitis are more neurovirulent in human neuronal culture and mouse models of HSV encephalitis, as compared to isolates collected from neonates with skin-limited disease. These findings suggest that inherent characteristics of the infecting HSV strain contribute to disease outcome following neonatal infection. [ABSTRACT FROM AUTHOR]

Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and RNA cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts that meet stringent criteria for expression. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORFs), six novel ORF-containing transcripts, and 15 transcripts encoding for messages that could alter protein functions through truncation or fusion of canonical ORFs. In addition, we detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking distinct gene transcription units. Among these chimeric proteins we detected an evolutionarily conserved protein containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies combined with mass spectrometry can reveal further complexity within viral transcriptomes and resulting proteomes. Author summary: Adenoviruses are important human pathogens that are also used as powerful tools for gene delivery and as vaccine agents. In addition, studies of the molecular biology of adenovirus have led to seminal discoveries in cell biology, RNA biology, and pathogenesis. While adenoviruses, have been exceptionally well-characterized over the past decades of research, much is still left to discover about their biology. By applying long-read RNA sequencing we have comprehensively reannotated the transcriptome of the Ad5 serotype. In doing so, we discovered an additional 35 abundant viral transcripts that potentially encode for 20 novel viral open reading frames. We demonstrate that one of these novel proteins joins two disparate genetic units into a chimeric protein that is evolutionarily conserved among adenoviruses. Discovery of this protein, E4orf6/DBP, recontextualizes prior studies on the function of the critical viral effector E4orf6. Furthermore, E4orf6/DBP is shown to modulate the morphology of membrane-less viral replication centers in the nucleus and function in cell-to-cell spread of the virus. Together, we showcase how modern techniques in RNA sequencing and mass spectrometry can reveal additional complexity in a well-studied model viral pathogen. [ABSTRACT FROM AUTHOR]

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]

Motivation The chemical modification of ribonucleotides regulates the structure, stability and interactions of RNAs. Profiling of these modifications using short-read (Illumina) sequencing techniques provides high sensitivity but low-to-medium resolution i.e. modifications cannot be assigned to specific transcript isoforms in regions of sequence overlap. An alternative strategy uses current fluctuations in nanopore-based long read direct RNA sequencing (DRS) to infer the location and identity of nucleotides that differ between two experimental conditions. While highly sensitive, these signal-level analyses require high-quality transcriptome annotations and thus are best suited to the study of model organisms. By contrast, the detection of RNA modifications in microbial organisms which typically have no or low-quality annotations requires an alternative strategy. Here, we demonstrate that signal fluctuations directly influence error rates during base-calling and thus provides an alternative approach for identifying modified nucleotides. Results DRUMMER (Detection of Ribonucleic acid Modifications Manifested in Error Rates) (i) utilizes a range of statistical tests and background noise correction to identify modified nucleotides with high confidence, (ii) operates with similar sensitivity to signal-level analysis approaches and (iii) correlates very well with orthogonal approaches. Using well-characterized DRS datasets supported by independent meRIP-Seq and miCLIP-Seq datasets we demonstrate that DRUMMER operates with high sensitivity and specificity. Availability and implementation DRUMMER is written in Python 3 and is available as open source in the GitHub repository: https://github.com/DepledgeLab/DRUMMER. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Macrophages play important roles in host immune defense against virus infection. During infection by herpes simplex virus 1 (HSV-1), macrophages acquire enhanced antiviral potential. Restriction of HSV-1 replication and progeny production is important to prevent viral spread, but the cellular mechanisms that inhibit the DNA virus in macrophages are unknown. SAMHD1 was recently identified as a retrovirus restriction factor highly expressed in macrophages. The SAMHD1 protein is expressed in both undifferentiated monocytes and differentiated macrophages, but retroviral restriction is limited to differentiated cells by modulation of SAMHD1 phosphorylation. It is proposed to block reverse transcription of retroviral RNA into DNA by depleting cellular deoxynucleotide triphosphates (dNTPs). Viruses with DNA genomes do not employ reverse transcription during infection, but replication of their viral genomes is also dependent on intracellular dNTP concentrations. Here, we demonstrate that SAMHD1 restricts replication of the HSV-1 DNA genome in differentiated macrophage cell lines. Depleting SAMHD1 in THP-1 cells enhanced HSV-1 replication, while ectopic overexpression of SAMHD1 in U937 cells repressed HSV-1 replication. SAMHD1 did not impact viral gene expression from incoming HSV-1 viral genomes. HSV-1 restriction involved the dNTP triphosphohydrolase activity of SAMHD1 and was partially overcome by addition of exogenous deoxynucleosides. Unlike retroviruses, restriction of HSV-1 was not affected by SAMHD1 phosphorylation status. Our results suggest that SAMHD1 functions broadly to inhibit replication of DNA viruses in nondividing macrophages. [ABSTRACT FROM AUTHOR]

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus). However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B) and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance. [ABSTRACT FROM AUTHOR]

In mammals, one of the most pronounced consequences of viral infection is the induction of type I interferons, cytokines with potent antiviral activity. Schlafen (Slfn) genes are a subset of interferon-stimulated early response genes (ISGs) that are also induced directly by pathogens via the interferon regulatory factor 3 (IRF3) pathway. However, many ISGs are of unknown or incompletely understood function. Here we show that human SLFN11 potently and specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 (HIV-1). Our study revealed that SLFN11 has no effect on the early steps of the retroviral infection cycle, including reverse transcription, integration and transcription. Rather, SLFN11 acts at the late stage of virus production by selectively inhibiting the expression of viral proteins in a codon-usage-dependent manner. We further find that SLFN11 binds transfer RNA, and counteracts changes in the tRNA pool elicited by the presence of HIV. Our studies identified a novel antiviral mechanism within the innate immune response, in which SLFN11 selectively inhibits viral protein synthesis in HIV-infected cells by means of codon-bias discrimination. [ABSTRACT FROM AUTHOR]

Adeno-associated virus type 2 (AAV2) is a human parvovirus that relies on a helper virus for efficient replication. Herpes simplex virus 1 (HSV-1) supplies helper functions and changes the environment of the cell to promote AAV2 replication. In this study, we examined the accumulation of cellular replication and repair proteins at viral replication compartments (RCs) and the influence of replicating AAV2 on HSV-1-induced DNA damage responses (DDR). We observed that the ATM kinase was activated in cells coinfected with AAV2 and HSV-1. We also found that phosphorylated ATR kinase and its cofactor ATR-interacting protein were recruited into AAV2 RCs, but ATR signaling was not activated. DNA-PKcs, another main kinase in the DDR, was degraded during HSV-1 infection in an ICP0-dependent manner, and this degradation was markedly delayed during AAV2 coinfection. Furthermore, we detected phosphorylation of DNA-PKcs during AAV2 but not HSV-1 replication. The AAV2-mediated delay in DNA-PKcs degradation affected signaling through downstream substrates. Overall, our results demonstrate that coinfection with HSV-1 and AAV2 provokes a cellular DDR which is distinct from that induced by HSV-1 alone. [ABSTRACT FROM AUTHOR]

The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex. [ABSTRACT FROM AUTHOR]

Virus infections have dramatic effects on structural and morphological characteristics of the host cell. The gene product of open reading frame 3 in the early region 4 (EAorf3) of adenovirus serotype 5 (Ad5) is involved in efficient replication and late protein synthesis. During infection with adenovirus mutants lacking the E4 region, the viral genomic DNA is joined into concatemers by cellular DNA repair factors, and this requires the Mre11/Rad50/Nbs1 complex. Concatemer formation can be prevented by the E4orf3 protein, which causes the cellular redistribution of the Mre11 complex. Here we show that E4orf3 colocalizes with components of the Mre11 complex in nuclear tracks and also in large cytoplasmic accumulations. Rearrangement of Mre11 and Rad50 by Ad5 E4orf3 is not dependent on interactions with Nbs1 or promyelocytic leukemia protein nuclear bodies. Late in infection the cytoplasmic inclusions appear as a distinct juxtanuclear accumulation at the centrosome and this requires an intact microtubule cytoskeleton. The large cytoplasmic accumulations meet the criteria defined for aggresomes, including γ-tubulin colocalization and formation of a surrounding vimentin cage. E4orf3 also appears to alter the solubility of the cellular Mre11 complex. These data suggest that E4orf3 can target the Mre11 complex to an aggresome and may explain how the cellular repair complex is inactivated during adenovirus infection. [ABSTRACT FROM AUTHOR]

• ICP0 is a viral E3 ubiquitin ligase that promotes HSV-1 infection. • ICP0 interacts with multiple component proteins of the ubiquitin pathway. • ICP0 disrupts multiple cellular processes activated in response to infection • ICP0 remodels the SUMO proteome to counteract host immune defences to infection. • ICP0 is an attractive drug target for the development of antiviral HSV-1 therapeutics. Herpes simplex virus 1 (HSV-1) hijacks ubiquitination machinery to modify the cellular proteome to create an environment permissive for virus replication. HSV-1 encodes its own RING-finger E3 ubiquitin (Ub) ligase, Infected Cell Protein 0 (ICP0), that directly interfaces with component proteins of the Ub pathway to inactivate host immune defences and cellular processes that restrict the progression of HSV-1 infection. Consequently, ICP0 plays a critical role in the infectious cycle of HSV-1 that is required to promote the efficient onset of lytic infection and productive reactivation of viral genomes from latency. This review will describe the current knowledge regarding the biochemical properties and known substrates of ICP0 during HSV-1 infection. We will highlight the gaps in the characterization of ICP0 function and propose future areas of research required to understand fully the biological properties of this important HSV-1 regulatory protein. [ABSTRACT FROM AUTHOR]

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.

Viruses exploit the host cell machinery to enable infection and propagation. This review discusses the complex landscape of DNA virus-host interactions, focusing primarily on herpesviruses and adenoviruses, which replicate in the nucleus of infected cells, and vaccinia virus, which replicates in the cytoplasm. We discuss experimental approaches used to discover and validate interactions of host proteins with viral genomes and how these interactions impact processes that occur during infection, including the host DNA damage response and viral genome replication, repair, and transcription. We highlight the current state of knowledge regarding virus-host protein interactions and also outline emerging areas and future directions for research.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.