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Background Variation in alternative tobacco product (ATP) constituents, heating potential, and consumer behaviors have made it difficult to characterize their health risks. To date, most toxicity studies of ATPs have used established cigarette endpoints to inform study design. Furthermore, to assess where ATPs fall on the tobacco harm continuum, with cigarettes representing maximum potential risk, studies have tended to compare the relative biological responses to ATPs against those due to cigarettes. Objectives 1) To characterize the exhalation profiles of two popular ATPs: electronic cigarettes (e-cigarettes) and hookah waterpipes (hookah) and 2) to determine if ATP exhalation patterns were representative of cigarette exhalation patterns. Methods Exhalation patterns were recorded (mouth only, nose only, or both mouth and nose) among individuals observed in the New York City tri-state area using a recognizable tobacco product (cigarette, e-cigarette, or hookah). Cigarette smokers and e-cigarette vapers were observed on city streets; water-pipe smokers were observed inside Manhattan hookah bars. Results E-cigarette vapers practiced exclusive nasal exhalation at far higher rates than did cigarette smokers (19.5% vs 4.9%). Among vapers, e-cigarette device type was also significantly associated with exhalation profile. Overall, cigarette smokers exhaled from their nose approximately half to one-third as often as ATP users (hookah and e-cigarettes, respectively). Conclusions Nasal exhalation of tobacco emissions appears to be a shared characteristic across several types of ATPs. It is therefore plausible that ATP-specific consumer behaviors may foster unique upper respiratory health consequences that have not been observed in smokers. Thus, product-specific behaviors should inform the prioritization of biological endpoints used in studies evaluating ATP toxicity and health effects.

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., (© 2024. The Author(s).)

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.

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 liquid-like 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., (© 2024. The Author(s).)

Importance: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells., Competing Interests: The authors declare no conflict of interest.

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.

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.

Biomolecular condensates formed by phase separation can compartmentalize and regulate cellular processes 1,2 . Emerging evidence has suggested that membraneless subcellular compartments in virus-infected cells form by phase separation 3-8 . Although linked to several viral processes 3-5,9,10 , evidence that phase separation contributes functionally to the assembly of progeny particles in infected cells is lacking. Here we show that phase separation of the human adenovirus 52-kDa protein has a critical role in the coordinated assembly of infectious progeny particles. We demonstrate that the 52-kDa protein is essential for the organization of viral structural proteins into biomolecular condensates. This organization regulates viral assembly such that capsid assembly is coordinated with the provision of viral genomes needed to produce complete packaged particles. We show that this function is governed by the molecular grammar of an intrinsically disordered region of the 52-kDa protein, and that failure to form condensates or to recruit viral factors that are critical for assembly results in failed packaging and assembly of only non-infectious particles. Our findings identify essential requirements for coordinated assembly of progeny particles and demonstrate that phase separation of a viral protein is critical for production of infectious progeny during adenovirus infection., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mutational signatures associated with apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)3 cytosine deaminase activity have been found in over half of cancer types, including some therapy-resistant and metastatic tumors. Driver mutations can occur in APOBEC3-favored sequence contexts, suggesting that mutagenesis by APOBEC3 enzymes may drive cancer evolution. The APOBEC3-mediated signatures are often detected in subclonal branches of tumor phylogenies and are acquired in cancer cell lines over long periods of time, indicating that APOBEC3 mutagenesis can be ongoing in cancer. Collectively, these and other observations have led to the proposal that APOBEC3 mutagenesis represents a disease-modifying process that could be inhibited to limit tumor heterogeneity, metastasis and drug resistance. However, critical aspects of APOBEC3 biology in cancer and in healthy tissues have not been clearly defined, limiting well-grounded predictions regarding the benefits of inhibiting APOBEC3 mutagenesis in different settings in cancer. We discuss the relevant mechanistic gaps and strategies to address them to investigate whether inhibiting APOBEC3 mutagenesis may confer clinical benefits in cancer., (© 2022. Springer Nature America, Inc.)

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., Competing Interests: The authors have declared that no competing interests exist.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy., (© 2022. The Author(s).)

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., (© The Author(s) 2022. Published by Oxford University Press.)

Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity., (© 2021 The Authors.)

Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. Adenovirus (AdV) encodes proteins from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteome changes induced by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, unbiased proteomics approach to analyze changes to the host proteome during infection with adenovirus serotype 5 (Ad5) virus. We used whole-cell proteomics to measure total protein abundances in the proteome during Ad5 infection. Since host antiviral proteins can antagonize viral infection by associating with viral genomes and inhibiting essential viral processes, we used Isolation of Proteins on Nascent DNA (iPOND) proteomics to identify proteins associated with viral genomes during infection with wild-type Ad5 or an E4 mutant virus. By integrating these proteomics data sets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. IMPORTANCE Viral infections induce myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, post-translational modifications, interactions, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We used an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner, suggesting ways that adenovirus counteracts potentially inhibitory host defenses. This study provides a global view of adenovirus-mediated proteome remodeling, which can serve as a model to investigate virus-host interactions of DNA viruses.

An unusual feature of papillomaviruses is that their genomes are packaged into virions along with host histones. Viral minichromosomes were visualized as "beads on a string" by electron microscopy in the 1970s but, to date, little is known about the posttranslational modifications of these histones. To investigate this, we analyzed the histone modifications in HPV16/18 quasivirions, wart-derived bovine papillomavirus (BPV1), and wart-derived human papillomavirus type 1 (HPV1) using quantitative mass spectrometry. The chromatin from all three virion samples had abundant posttranslational modifications (acetylation, methylation, and phosphorylation). These histone modifications were verified by acid urea polyacrylamide electrophoresis and immunoblot analysis. Compared to matched host cell controls, the virion minichromosome was enriched in histone modifications associated with active chromatin and depleted for those commonly found in repressed chromatin. We propose that the viral minichromosome acquires specific histone modifications late in infection that are coupled to the mechanisms of viral replication, late gene expression, and encapsidation. We predict that, in turn, these same modifications benefit early stages of infection by helping to evade detection, promoting localization of the viral chromosome to beneficial regions of the nucleus, and promoting early transcription and replication. IMPORTANCE A relatively unique feature of papillomaviruses is that the viral genome is associated with host histones inside the virion. However, little is known about the nature of the epigenome within papillomavirions or its biological relevance to the infectious viral cycle. Here, we define the epigenetic signature of the H3 and H4 histones from HPV16 virions generated in cell culture and native human papillomavirus type 1 (HPV1) and bovine papillomavirus 1 (BPV1) virions isolated from bovine and human wart tissue. We show that native virions are enriched in posttranslational modifications associated with active chromatin and depleted with those associated with repressed chromatin compared to cellular chromatin. Native virions were also enriched in the histone variant H3.3 compared to the canonical histone H3.1. We propose that the composition of virion-packaged chromatin reflects the late stages of the viral life cycle and promotes the early stages of infection by being primed for viral transcription.

Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.

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 (m 6 A) to mRNAs. While m 6 A-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 m 6 A mapping using short-read sequencing, here we profile m 6 A within the adenovirus transcriptome using a combination of meRIP-seq and direct RNA long-read sequencing to yield both nucleotide and transcript-resolved m 6 A detection. Although both early and late viral transcripts contain m 6 A, depletion of m 6 A writer METTL3 specifically impacts viral late transcripts by reducing their splicing efficiency. These data showcase a new technique for m 6 A discovery within individual transcripts at nucleotide resolution, and highlight the role of m 6 A in regulating splicing of a viral pathogen.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

Paramyxoviruses are negative-sense single-stranded RNA viruses that comprise many important human and animal pathogens, including human parainfluenza viruses. These viruses bud from the plasma membrane of infected cells after the viral ribonucleoprotein complex (vRNP) is transported from the cytoplasm to the cell membrane via Rab11a-marked recycling endosomes. The viral proteins that are critical for mediating this important initial step in viral assembly are unknown. Here, we used the model paramyxovirus, murine parainfluenza virus 1, or Sendai virus (SeV), to investigate the roles of viral proteins in Rab11a-driven virion assembly. We previously reported that infection with SeV containing high levels of copy-back defective viral genomes (DVGs) (DVG-high SeV) generates heterogenous populations of cells. Cells enriched in full-length (FL) virus produce viral particles containing standard or defective viral genomes, while cells enriched in DVGs do not, despite high levels of defective viral genome replication. Here, we took advantage of this heterogenous cell phenotype to identify proteins that mediate interaction of vRNPs with Rab11a. We examined the roles of matrix protein and nucleoprotein and determined that their presence is not sufficient to drive interaction of vRNPs with recycling endosomes. Using a combination of mass spectrometry and comparative analyses of protein abundance and localization in DVG-high and FL-virus-high (FL-high) cells, we identified viral polymerase complex component protein L and, specifically, its cofactor C as interactors with Rab11a. We found that accumulation of L and C proteins within the cell is the defining feature that differentiates cells that proceed to viral egress from cells containing viruses that remain in replication phases. IMPORTANCE Paramyxoviruses are members of a family of viruses that include a number of pathogens imposing significant burdens on human health. In particular, human parainfluenza viruses are an important cause of pneumonia and bronchiolitis in children for which there are no vaccines or directly acting antivirals. These cytoplasmic replicating viruses bud from the plasma membrane and co-opt cellular endosomal recycling pathways to traffic viral ribonucleoprotein complexes from the cytoplasm to the membrane of infected cells. The viral proteins required for viral engagement with the recycling endosome pathway are still not known. Here, we used the model paramyxovirus Sendai virus, or murine parainfluenza virus 1, to investigate the role of viral proteins in this initial step of viral assembly. We found that the viral polymerase components large protein L and accessory protein C are necessary for engagement with recycling endosomes. These findings are important in identifying viral proteins as potential targets for development of antivirals., (Copyright © 2020 Genoyer et al.)

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., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Recombinant adeno-associated virus has emerged as one of the most promising gene therapy delivery vectors. Development of these vectors took advantage of key features of the wild-type adeno-associated virus (AAV), enabled by basic studies of the underlying biology and requirements for transcription, replication, and packaging of the viral genome. Each step in generating and utilizing viral vectors involves numerous molecular interactions that together determine the efficiency of vector production and gene delivery. Once delivered into the cell, interactions with host proteins will determine the fate of the viral genome, and these will impact the intended goal of gene delivery. Here, we provide an overview of known interactions of the AAV genome with viral and cellular proteins involved in its amplification, packaging, and expression. Further appreciation of how the AAV genome interacts with host factors will enhance how this simple virus can be harnessed for an array of vector purposes that benefit human health.

Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy. See related commentary by Green and Neelapu, p. 492 ., (©2020 American Association for Cancer Research.)

DNA viruses that replicate in the nucleus encompass a range of ubiquitous and clinically important viruses, from acute pathogens to persistent tumor viruses. These viruses must co-opt nuclear processes for the benefit of the virus, whilst evading host processes that would otherwise attenuate viral replication. Accordingly, DNA viruses induce the formation of membraneless assemblies termed viral replication compartments (VRCs). These compartments facilitate the spatial organization of viral processes and regulate virus-host interactions. Here, we review advances in our understanding of VRCs. We cover their initiation and formation, their function as the sites of viral processes, and aspects of their composition and organization. In doing so, we highlight ongoing and emerging areas of research highly pertinent to our understanding of nuclear-replicating DNA viruses., Competing Interests: The authors declare no conflict of interest.

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.

Adenoviruses represent ubiquitous and clinically significant human pathogens, gene-delivery vectors, and oncolytic agents. The study of adenovirus-infected cells has long been used as an excellent model to investigate fundamental aspects of both DNA virus infection and cellular biology. While many key details supporting a well-established model of adenovirus replication have been elucidated over a period spanning several decades, more recent findings suggest that we have only started to appreciate the complex interplay between viral genome replication and cellular processes. Here, we present a concise overview of adenovirus DNA replication, including the biochemical process of replication, the spatial organization of replication within the host cell nucleus, and insights into the complex plethora of virus-host interactions that influence viral genome replication. Finally, we identify emerging areas of research relating to the replication of adenovirus genomes., (© 2019 Federation of European Biochemical Societies.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Homologous recombination (HR) is considered a major driving force of evolution because it generates and expands genetic diversity. Evidence of HR between coinfecting herpesvirus DNA genomes can be found frequently both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when coinfecting viral genomes interact. To study the spatiotemporal requirements for intergenomic recombination, we developed an assay with dual-color FISH that enables detection of HR between different pairs of coinfecting HSV-1 genomes. Our results revealed that HR increases intermingling of RCs derived from different genomes. Furthermore, inhibition of RC movement reduces the rate of HR events among coinfecting viruses. Finally, we observed correlation between nuclear size and the number of RCs per nucleus. Our findings suggest that both viral replication and recombination are subject to nuclear spatial constraints. Other DNA viruses and cellular DNA are likely to encounter similar restrictions.-Tomer, E., Cohen, E. M., Drayman, N., Afriat, A., Weitzman, M. D., Zaritsky, A., Kobiler, O. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.

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., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors have declared that no competing interests exist.

More than 14,000 neonates are infected with herpes simplex virus (HSV) annually. Approximately half display manifestations limited to the skin, eyes, or mouth (SEM disease). The rest develop invasive infections that spread to the central nervous system (CNS disease or encephalitis) or throughout the infected neonate (disseminated disease). Invasive HSV disease is associated with significant morbidity and mortality, but the viral and host factors that predispose neonates to these forms are unknown. To define viral diversity within the infected neonatal population, we evaluated 10 HSV-2 isolates from newborns with a range of clinical presentations. To assess viral fitness independently of host immune factors, we measured viral growth characteristics in cultured cells and found diverse in vitro phenotypes. Isolates from neonates with CNS disease were associated with larger plaque size and enhanced spread, with the isolates from cerebrospinal fluid (CSF) exhibiting the most robust growth. We sequenced complete viral genomes of all 10 neonatal viruses, providing new insights into HSV-2 genomic diversity in this clinical setting. We found extensive interhost and intrahost genomic diversity throughout the viral genome, including amino acid differences in more than 90% of the viral proteome. The genes encoding glycoprotein G (gG; US4), glycoprotein I (gI; US7), and glycoprotein K (gK; UL53) and viral proteins UL8, UL20, UL24, and US2 contained variants that were found in association with CNS isolates. Many of these viral proteins are known to contribute to cell spread and neurovirulence in mouse models of CNS disease. This report represents the first application of comparative pathogen genomics to neonatal HSV disease. IMPORTANCE Herpes simplex virus (HSV) causes invasive disease in half of infected neonates, resulting in significant mortality and permanent cognitive morbidity. The factors that contribute to invasive disease are not understood. This study revealed diversity among HSV isolates from infected neonates and detected the first associations between viral genetic variations and clinical disease manifestations. We found that viruses isolated from newborns with encephalitis showed enhanced spread in culture. These viruses contained protein-coding variations not found in viruses causing noninvasive disease. Many of these variations were found in proteins known to impact neurovirulence and viral spread between cells. This work advances our understanding of HSV diversity in the neonatal population and how it may impact disease outcome., (Copyright © 2019 Akhtar et al.)

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 dl1004, 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., (Copyright © 2018. Published by Elsevier B.V.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Viral DNA genomes have limited coding capacity and therefore harness cellular factors to facilitate replication of their genomes and generate progeny virions. Studies of viruses and how they interact with cellular processes have historically provided seminal insights into basic biology and disease mechanisms. The replicative life cycles of many DNA viruses have been shown to engage components of the host DNA damage and repair machinery. Viruses have evolved numerous strategies to navigate the cellular DNA damage response. By hijacking and manipulating cellular replication and repair processes, DNA viruses can selectively harness or abrogate distinct components of the cellular machinery to complete their life cycles. Here, we highlight consequences for viral replication and host genome integrity during the dynamic interactions between virus and host.

During viral replication in the nucleus, the DNA genomes of adenoviruses are accessible to cellular DNA-binding proteins. Human adenovirus type 5 (Ad5) targets the cellular Mre11-Rad50-Nbs1 complex (MRN) to evade detection by the DNA damage response (DDR). Ad5 mutants that cannot target MRN have reduced viral propagation. Previous studies showed that diverse adenovirus serotypes interact differently with MRN. While these studies revealed diverse MRN interactions among serotypes, it remains unclear how these differences influence viral replication. Here, we examined effects of the DDR on several adenovirus serotypes. We demonstrate that wild-type Ad9 and Ad12 do not overcome MRN impairment. We also examined viral proteins involved in targeting MRN and found that unlike Ad5-E4orf3, expression of Ad9-E4orf3 is not sufficient for MRN mislocalization observed during infection. We conclude that adenovirus serotypes target MRN in distinct ways, and the MRN complex can impair DNA replication of wild-type viruses across the adenovirus family., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Viral DNA genomes replicating in cells encounter a myriad of host factors that facilitate or hinder viral replication. Viral proteins expressed early during infection modulate host factors interacting with viral genomes, recruiting proteins to promote viral replication, and limiting access to antiviral repressors. Although some host factors manipulated by viruses have been identified, we have limited knowledge of pathways exploited during infection and how these differ between viruses. To identify cellular processes manipulated during viral replication, we defined proteomes associated with viral genomes during infection with adenovirus, herpes simplex virus and vaccinia virus. We compared enrichment of host factors between virus proteomes and confirmed association with viral genomes and replication compartments. Using adenovirus as an illustrative example, we uncovered host factors deactivated by early viral proteins, and identified a subgroup of nucleolar proteins that aid virus replication. Our data sets provide valuable resources of virus-host interactions that affect proteins on viral genomes., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Tobacco use continues to be the leading cause of preventable death worldwide., Objective: To assess perceived tobacco educational preparation of undergraduate and graduate nurses in a large urban university., Design: A cross-sectional web-based survey of all nursing students in an urban college of nursing was conducted to assess perceived tobacco educational content, skills, and self-efficacy for cessation counseling with patients who smoke cigarettes and use alternative tobacco products (e.g., hookah and e-cigarettes)., Results: Participants reported the most education about health effects of tobacco use (67%), and less than 6% reported education about alternative tobacco products. While the majority of nurses agreed that advising patients to quit is a priority, less than 40% reported receiving sufficient training in cessation counseling., Conclusions: There continues to be a compelling need articulated by leaders in tobacco policy and research over the past decade for a more vigorous response by nurses to the tobacco epidemic.

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.This article is part of the themed issue 'Human oncogenic viruses'., (© 2017 The Author(s).)

Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin. IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases, such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally downregulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome., (Copyright © 2017 American Society for Microbiology.)

Mutational signatures in cancer genomes have implicated the APOBEC3 cytosine deaminases in oncogenesis, possibly offering a therapeutic vulnerability. Elevated APOBEC3B expression has been detected in solid tumors, but expression of APOBEC3A (A3A) in cancer has not been described to date. Here, we report that A3A is highly expressed in subsets of pediatric and adult acute myelogenous leukemia (AML). We modeled A3A expression in the THP1 AML cell line by introducing an inducible A3A gene. A3A expression caused ATR-dependent phosphorylation of Chk1 and cell-cycle arrest, consistent with replication checkpoint activation. Further, replication checkpoint blockade via small-molecule inhibition of ATR kinase in cells expressing A3A led to apoptosis and cell death. Although DNA damage checkpoints are broadly activated in response to A3A activity, synthetic lethality was specific to ATR signaling via Chk1 and did not occur with ATM inhibition. Our findings identify elevation of A3A expression in AML cells, enabling apoptotic sensitivity to inhibitors of the DNA replication checkpoint and suggesting it as a candidate biomarker for ATR inhibitor therapy. Cancer Res; 77(17); 4579-88. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Infection with herpes simplex virus-1 (HSV-1) brings numerous changes in cellular gene expression. Levels of most host mRNAs are reduced, limiting synthesis of host proteins, especially those involved in antiviral defenses. The impact of HSV-1 on host microRNAs (miRNAs), an extensive network of short non-coding RNAs that regulate mRNA stability/translation, remains largely unexplored. Here we show that transcription of the miR-183 cluster (miR-183, miR-96, and miR-182) is selectively induced by HSV-1 during productive infection of primary fibroblasts and neurons. ICP0, a viral E3 ubiquitin ligase expressed as an immediate-early protein, is both necessary and sufficient for this induction. Nuclear exclusion of ICP0 or removal of the RING (really interesting new gene) finger domain that is required for E3 ligase activity prevents induction. ICP0 promotes the degradation of numerous host proteins and for the most part, the downstream consequences are unknown. Induction of the miR-183 cluster can be mimicked by depletion of host transcriptional repressors zinc finger E-box binding homeobox 1 (ZEB1)/-crystallin enhancer binding factor 1 (δEF1) and zinc finger E-box binding homeobox 2 (ZEB2)/Smad-interacting protein 1 (SIP1), which we establish as new substrates for ICP0-mediated degradation. Thus, HSV-1 selectively stimulates expression of the miR-183 cluster by ICP0-mediated degradation of ZEB transcriptional repressors., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.