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This study provides a genome-wide analysis of changes in promoter-proximal polymerase II (Pol II) pausing on host genes induced by HSV-1 infection. It shows that standard measures of pausing, i.e., pausing indices, do not properly capture the complex and unsuspected alterations in Pol II pausing occurring in HSV-1 infection.

Nucleoside analogues like 4-thiouridine (4sU) are used to metabolically label newly synthesized RNA. Chemical conversion of 4sU before sequencing induces T-to-C mismatches in reads sequenced from labelled RNA, allowing to obtain total and labelled RNA expression profiles from a single sequencing library. Cytotoxicity due to extended periods of labelling or high 4sU concentrations has been described, but the effects of extensive 4sU labelling on expression estimates from nucleotide conversion RNA-seq have not been studied. Here, we performed nucleotide conversion RNA-seq with escalating doses of 4sU with short-term labelling (1h) and over a progressive time course (up to 2h) in different cell lines. With high concentrations or at later time points, expression estimates were biased in an RNA half-life dependent manner. We show that bias arose by a combination of reduced mappability of reads carrying multiple conversions, and a global, unspecific underrepresentation of labelled RNA due to impaired reverse transcription efficiency and potentially global reduction of RNA synthesis. We developed a computational tool to rescue unmappable reads, which performed favourably compared to previous read mappers, and a statistical method, which could fully remove remaining bias. All methods developed here are freely available as part of our GRAND-SLAM pipeline and grandR package.

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35’s function. Using electrophoretic mobility shift assays (EMSA), we demonstrate that purified M35 protein specifically binds to the regulatory DNA element that governs transcription of the first type I IFN gene induced in non-immune cells,Ifnb1. Determination of M35’s crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35’s immunomodulatory activity. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signalling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the hostIfnb1promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signalling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labelled transcripts (SLAM-seq), and assessed M35’s global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically down-regulated basal expression of IRF3-dependent genes, and during MCMV infection, M35 impaired expression of IRF3-responsive genes aside ofIfnb1. Our results suggest that M35-DNA binding directly antagonises gene induction by IRF3 and impairs the antiviral response more broadly than formerly recognised.ImportanceReplication of the ubiquitous human cytomegalovirus (CMV) in healthy individuals mostly goes unnoticed, but can impair foetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes. Unrelated proteins from other herpesviruses employ the same mechanism, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3′ untranslated region of the major viral immediate early 3 ( ) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection . Furthermore, no differences in the induction of virus-specific CD8 T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes.

Herpes simplex virus 1 (HSV-1) infection exerts a profound shut-off of host gene expression at multiple levels. Recently, HSV-1 infection was reported to also impact promoter-proximal RNA Polymerase II (Pol II) pausing, a key step in the eukaryotic transcription cycle, with decreased and increased Pol II pausing observed for activated and repressed genes, respectively. Here, we demonstrate that HSV-1 infection induces more complex alterations in promoter-proximal pausing than previously suspected for the vast majority of cellular genes. While pausing is generally retained, it is shifted to more downstream and less well-positioned sites for most host genes. We exclude that this is an artefact of alternativede novotranscription initiation at downstream sites or read-in transcription originating from disruption of transcription termination for upstream genes. Use of downstream 2ndpause sites associated with +1 nucleosomes was previously observed upon NELF depletion. However, downstream shifts of Pol II pausing upon HSV-1 infection are much more pronounced than observed upon NELF depletion. Thus, our study reveals a novel aspect in which HSV-1 infection fundamentally reshapes host transcriptional processes with implications for our understanding of maintenance of promoter-proximal Pol II pausing in eukaryotic cells.

In the last decade Magnetic nanoparticles (MNPs) have gained an enormous interest in specialized areas such as medicine, cancer theranostics, biosensing, catalysis, agriculture, and the environmental protection. By controlled engineering of specific surface properties, named functionalization, MNPs are gaining special features for desired applications, e.g., bioassays for the detection of biomolecules or biomarkers such as antibodies. The characterization as well as a highly specific measurement of such binding states is of high interest and limited to highly sensitive techniques such as ELISA (Enzyme-linked Immunosorbent Assay) or flow cytometry, which are relatively inflexible, difficult to handle, expensive and time-consuming. Novel upcoming methods, such as ACS (AC susceptometry) or MPS (Magnetic Particle Spectroscopy), exploit the magnetization response of functionalized MNP ensembles to assess specific information about the MNP mobility within their environment as well as the conjugations of chemical or biological compounds on their surface. Both methods have shown promising results reaching similar sensitivities within short measurement times but showing difficulties in data interpretation. Here, we report a novel method, COMPASS (Critical Offset Magnetic PArticle SpectroScopy), which is based on a critical offset magnetic field of MNPs, which enables sensitive detection to minimal changes in mobility of MNP ensembles, e.g., resulting from SARS-CoV-2 antibodies binding to the S antigen on the surface of functionalized MNPs. With a validated sensitivity of 0.85 fmole/50 µl sample volume ( ≙ 33 pM) SARS-CoV-2-S1 antibodies, measured with a low-cost portable COMPASS device, the proposed technique is not only competitive with the sensitivity of commonly used ELISA or flow cytometry methods but provides more flexibility, robustness and rapid measurement withinwell below a minute per sample, including sample conjugation, mixing and incubation times. The underlying physical effect is based on an offset magnetic field induced suppression of a higher harmonic in the nonlinear magnetization response of the MNP to a time varying magnetic field resulting in a highly sensitive response of the signal phase to minimal changes in particle mobility. Since this effect is independent of MNP concentration, the sample handling is much simpler and robust. Our method thus may pave the way for deeper insights into complex and rapid binding dynamics of functionalization chemistry and can lead to a huge step forwards in point-of-care diagnostics as well as impacts other fields in research and industries.

Glaciers in the Himalaya–Karakoram mountain ranges harbour approximately half of the ice volume in High-mountain Asia and modulate the flow of freshwater to almost 869 million people within the Indus, Tarim, Ganges and Brahmaputra river basins. Since the mid-twentieth century, rising temperatures have led to unsustainably high melting rates for many glaciers, particularly in the Himalaya, temporarily increasing summer meltwater run-off but continuously reducing the ice-storage volume. In this Review, we discuss how and why glaciers and meltwater supplies have changed, how they will likely evolve in the future and how these changes impact water resources and water-related hazards. Heterogeneous glacier retreat is changing streamflow patterns, in turn, affecting the incidence of glacial-lake outburst floods and exacerbating the risk of flooding and water shortages associated with future climate change. These changes could negatively impact downstream populations and infrastructure, including the thriving hydropower sector and some of the world’s largest irrigated agriculture systems, by making water flow more extreme and unpredictable. An improved in situ monitoring network for weather, hydrology and glacier change is a crucial requirement for predicting the future of this resource and associated hazards, and their impact on regional water, energy and food security. Glaciers in the Himalaya and Karakoram mountain ranges provide freshwater and hydropower to millions of people but are also melting at unsustainably high rates. This Review discusses recent and projected changes in glacier melt and resulting implications for regional water-related hazards and water resources.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2., Interactions between SARS-CoV-2 viral RNAs and host cell proteins during infection are evaluated to improve our understanding of viral RNA functions and the host innate immune response.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation

Herpes simplex virus 1 (HSV-1) infection and stress responses disrupt transcription termination by RNA Polymerase II (Pol II). In HSV-1 infection but not upon salt or heat stress, this is accompanied by a dramatic increase in chromatin accessibility downstream of genes. Here, we show that the HSV-1 immediate-early protein ICP22 is both necessary and sufficient to induce downstream open chromatin regions (dOCRs) upon disrupted transcription termination. This was matched by a marked ICP22-dependent loss of histones downstream of affected genes consistent with impaired histone repositioning in the wake of Pol II. Efficient knock-down of the ICP22-interacting histone chaperon FACT, though insufficient to induce dOCRs in ΔICP22 infection, increased dOCR induction upon wild-type HSV-1 infection. Interestingly, this was accompanied by a marked increase in chromatin accessibility within gene bodies. We propose a model in which allosteric changes in Pol II composition downstream of genes and ICP22-mediated interference with FACT activity explain the differential impairment of histone re-positioning in the wake of Pol II observed in HSV-1 infection.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a novel cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the latent-lytic switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture, which impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 was sufficient to trigger virus reactivation from latency thereby identifying it as a readily drugable master regulator of the herpesvirus latent-lytic switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders like myalgic encephalitis/chronic fatigue syndrome (ME/CFS) and Long-COVID.

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2ccs), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell lines carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2ccs debulking that favors cell survival and virus production., Author summary The N-terminal domains of the herpesvirus large tegument proteins encode a conserved cysteine protease with ubiquitin- and NEDD8-specific deconjugase activity. Members of the viral enzyme family regulate different aspects of the virus life cycle including virus replication, the assembly of infectious virus particles and the host innate anti-viral response. However, only few substrates have been validated under physiological conditions of expression and very little is known on the mechanisms by which the enzymes contribute to the reprograming of cellular functions that are required for efficient infection and virus production. Cellular type I and type II topoisomerases (TOP1 and TOP2) resolve topological problems that arise during DNA replication and transcription and are therefore essential for herpesvirus replication. We report that the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1 selectively regulates the activity of TOP2 in cells treated with the TOP2 poison Etoposide and during productive infection. Using transiently transfected and stable cell lines that express catalytically active or inactive BPLF1, we found that BPLF1 interacts with both TOP2α and TOP2β in co-immunoprecipitation and in vitro pull-down assays and the active enzyme stabilizes TOP2 trapped in TOP2ccs, promoting a shift towards TOP2 SUMOylation. This hinders the activation of DNA-damage responses and reduces the toxicity of Etoposide. The physiological relevance of this finding was validated using pairs of EBV carrying HEK-293T cells and EBV immortalized lymphoblastoid cell lines (LCLs) expressing the wild type or catalytic mutant enzyme. Using knockout LCLs we found that the capacity of BPLF1 to rescue of Etoposide toxicity is dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair.

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonizes several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is less well understood. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, ZAP-S and ZAP-L, is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP results in reduced viral mRNA and protein levels and decelerates the progression of HCMV infection. Metabolic RNA labeling combined with high-throughput sequencing (SLAM-seq) revealed that most of the gene expression changes late in infection result from the general attenuation of HCMV. Furthermore, at early stages of infection, ZAP restricts HCMV by destabilizing a distinct subset of viral mRNAs, particularly those from the previously uncharacterized UL4-UL6 HCMV gene locus. Through enhanced cross-linking immunoprecipitation and sequencing analysis (eCLIP-seq), we identified the transcripts expressed from this HCMV locus as the direct targets of ZAP. Moreover, our data show that ZAP preferentially recognizes not only CG, but also other cytosine-rich sequences, thereby expanding its target specificity. In summary, this report is the first to reveal direct targets of ZAP during HCMV infection, which strongly indicates that transcripts from the UL4-UL6 locus may play an important role for HCMV replication.IMPORTANCE Viral infections have a large impact on society, leading to major human and economic losses and even global instability. So far, many viral infections, including human cytomegalovirus (HCMV) infection, are treated with a small repertoire of drugs, often accompanied by the occurrence of resistant mutants. There is no licensed HCMV vaccine in sight to protect those most at risk, particularly immunocompromised individuals or pregnant women who might otherwise transmit the virus to the fetus. Thus, the identification of novel intervention strategies is urgently required. In this study, we show that ZAP decelerates the viral gene expression cascade, presumably by selectively handpicking a distinct set of viral transcripts for degradation. Our study illustrates the potent role of ZAP as an HCMV restriction factor and sheds light on a possible role for UL4 and/or UL5 early during infection, paving a new avenue for the exploration of potential targets for novel therapies.

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2α and TOP2β in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2βcc, enhanced TOP2β SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.

The conserved, structural HSV-1 tegument protein pUL36 is essential for both virus entry and assembly. While its N-terminal de-ubiquitinase (DUB) activity is dispensable for infection in cell culture, it is required for efficient virus spread in vivo by acting as a potent viral immune evasin. Here, we show that the pUL36 DUB activity was required to overcome interferon-(IFN)-mediated suppression of both plaque initiation and progression to productive infection. Immediately upon virus entry, incoming tegument-derived pUL36-DUB activity helped the virus to escape intrinsic antiviral resistance and efficiently initiate lytic virus replication in IFN-primed cells. Subsequently, de novo expressed pUL36-DUB augmented the efficiency of productive infection and virus yield. Interestingly, removal of IFN shortly after inoculation only resulted in a partial rescue of plaque formation, indicating that an IFN-induced defense mechanism eliminates invading virus particles unless counteracted by pUL36-DUB activity. Taken together, we demonstrated that the pUL36 DUB disarms IFN-induced antiviral responses at two levels, namely, to protect the infectivity of invading virus as well as to augment productive virus replication in IFN-primed cells.Author SummaryHSV-1 is an ubiquitous human pathogen that is responsible for common cold sores but may also cause life-threatening disease. pUL36 is an essential and conserved protein of infectious herpesvirus virions with a unique de-ubiquitinating (DUB) activity. The pUL36 DUB is dispensable for efficient virus infection in cell culture but represents an important viral immune evasin in vivo. Here, we showed that tegument-derived DUB activity delivered by the invading virus particles is required to overcome IFN-induced host resistance and to initiate efficient lytic infection. De novo expressed pUL36 DUB subsequently augments productive infection and virus yield. These data indicate that the pUL36 DUB antagonizes the activity of yet unidentified IFN-inducible E3 ligases to facilitate productive infection at multiple levels. Our findings underscore the therapeutic potential of targeting conserved herpesvirus DUBs to prevent or treat herpesvirus disease.

Southwest China plays a key role in global hydropower; its current installed capacity is roughly equal to that of the rest of Asia. This immense hydropower capacity stems from some of the world’s largest dams as well as the world’s greatest density of smaller projects. However, our knowledge about the region’s hydropower utilisation patterns and spatio-temporal impacts is based on limited empirical data. This chapter provides a detailed analysis of the uneven spatio-temporal hydropower development in the area and the fluctuating oversupply of hydroelectricity. While as of 2019 hydropower development is advanced in Sichuan (77 GW, 316 TWh) and Yunnan (64 GW, 270 TWh), it is still in an early stage in Tibet. However, Tibet’s hydropower development might even surpass both provinces in the future. The second part of the chapter analyses the utilisation of hydroelectricity, with an emphasis on Yunnan. Our analysis focuses on the trade-offs (benefits, disadvantages, and current trends) related to large-scale power exports to coastal load centres, mapping out these exports for all of Southwest China. Further, we discuss the unique role of power-intensive industries within Yunnan and the impacts of rural electrification on local utilisation of hydropower.

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation, a nuclear export defect and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.

The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes., Herpes simplex virus 1 (HSV-1) induces a profound host shutoff during lytic infection. The virion host shutoff (vhs) protein plays a key role in this process by efficiently cleaving host and viral mRNAs. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in host transcriptional activity. To dissect relative contributions of both mechanisms and elucidate gene-specific host transcriptional responses throughout the first 8 h of lytic HSV-1 infection, we used transcriptome sequencing of total, newly transcribed (4sU-labeled) and chromatin-associated RNA in wild-type (WT) and Δvhs mutant infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8 h postinfection (p.i.). In parallel, host transcriptional activity dropped to 10 to 20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA and was surprisingly concordant between WT and Δvhs infections. Both induced strong transcriptional upregulation of a small subset of genes that were poorly expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional downregulation of at least 150 cellular genes, in particular of many integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8 h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8 h of lytic HSV-1 infection. IMPORTANCE The HSV-1 virion host shutoff (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity, as well as virus-induced global loss of host transcriptional activity, during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were dominated by these two global effects. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infections, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection, and identified vhs-dependent transcriptional downregulation of the integrin adhesome and extracellular matrix components. The latter explained seemingly gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8 h p.i. for many of the respective genes.

Axial cracking inspections have become common place on a global level within pipeline operator’s integrity management programs. As technology continues to improve, operators are presented with more accurate assessments of the assets that are in current operation. However as more information is collected more threats are being identified and need to be assessed in a manner that is more applicable to their specific morphology. It is well known that vintage ERW manufacturing techniques can suffer from a wide range of potential threats such as lack of fusion or inclusions within the steel forming hook cracks during the rolling and welding process. Current In-line inspection technologies that are designed to detect, Identify and size cracklike flaws in pipelines are very proficient at doing so. However, due to the physical principals of the Ultrasonic pulse echo technology, deep features approaching, or above pulse echo saturation amplitudes pose challenges in determining accurate depth sizing. In 2015 a Canadian pipeline operator determined the need to inspect one of their 16” assets for axial crack-like indications. During the analysis of this inspection data set, a number of saturated crack-like indications were reported. Saturated cracklike signals present a challenge to operators as they have to be considered in a conservative manner as 4mm or deeper which in turn leads to difficulties in the prioritization of resources associated with the excavation program. The operator approached NDT Global in 2017, after the release of NDT Global’s Enhanced sizing depth algorithm to reevaluate the features that were present in the previous crack inspection data set. Working together with the operator, NDT Global applied the Enhanced sizing methodology to all features of significance in the pipeline segment and compared the results to lab measurements and in field NDE measurements. The outcome of the reanalysis using the most up to date software algorithms utilizing enhanced sizing showed great benefits by increasing the accuracy of the crack depth sizing as NDT Global was now able to report full through wall depth sizing, however there were still some limitations on the ability to accurately size crack-like features as the primary threat is believed to be a result of hook cracks. As a final step in this program NDT Global was provided sample spools that were cut out of the pipeline segment to perform a pull testing campaign utilizing the newest crack detection technology that was specifically targeted towards accurately sizing tilted and skewed crack like features. The authors will briefly discuss the pipeline system and inspection campaign and in detail will discuss the benefits of using technology that has been developed to help pipeline operators better understand the threats in their integrity management program.

Pipeline operators use different approaches, often in combination, to ensure the safe operation of an asset. Historically, hydrostatic testing is the most accepted methodology for assessing critical flaws in a pipeline system, achieved by stressing the pipeline above the standard operating limits. By design, a release during this test removes a critical flaw from the system. There are significant drawbacks to this type of assessment. Such drawbacks include high costs of implementation, feature growth, previously blunt defects sharpening and system downtime. Driven by the operator (Marathon Pipe Line) to investigate alternative approaches, a consortium of (4) parties formed to develop and validate an alternative and enhanced solution. The research and execution of the project was structured in (4) phases. • Phase I — Determine which crack morphologies are challenging to ILI technology in terms of detection and depth determination and test initial improvements to ILI technology. This included loop testing improved crack detection ILI robots and destructive assessment with pipe samples to identify gaps in ILI technology and analysis techniques. • Phase II — Assess vendor improved ILI technology on the operating pipelines using advanced in the ditch non-destructive evaluation (NDE) methods and destructive testing of pipe samples from the pipelines. Identify the additional improvements if necessary. • Phase III — Implement specific improvements to ILI technology that are completed based on Phase I and II results. Validate the new ILI platform on the specific pipeline. • Phase IV — Determination whether the new ILI platform can provide equivalent pipe seam system reliability to hydrostatic testing. A probabilistic approach will be used that would account for the expected statistical distribution of pipe properties, fatigue crack growth rates, position along the pipeline, and flaw sizes. This paper is a summary of the research and work for this ILI In Lieu of a Hydrostatic Testing initiative.

Cracks in dents or linear anomalies interacting with dents are a major pipeline threat. These combined anomalies represent challenges to the Mechanical Engineers that design ILI tools as they need to keep the sensor in an optimal position towards the inner pipe wall. Ultrasonic Crack (UC) tools consist in a sensor plate with a fixed incidence angle that depends on the coupling medium. This plate is then attached to the skids; these are in constant contact with the internal pipe wall. When the tool interacts with a dent, the incidence angle is not optimal; therefore, detection of any interacting feature is compromised. By not having the optimum angles in the pipe wall, the amplitudes from the reflections caused by cracks will be attenuated. Depending on the magnitude of the attenuation, these might be below analysis thresholds meaning that an algorithm and/or analyst will not consider them as relevant signals. Up to this point, detection of interacting features sounds like a “guess “ or “luck”. So, how can we use UC inspection to detect the interacting features? How can operators manage their assets knowing that they have dents but there is an uncertainty if there are interacting features? To answer these questions, a systematic approach had to be used. It consisted of multiple phases where 1.- The mechanical design of the tool was understood, 2.- Simulation campaigns to understand the ultrasonic pulse while interacting with the dent, 3.- Pump tests with artificial features, and 4.- Pump test with real features. All of the data gathered through the different phases allowed the authors to understand the attributes from the features and conditions that influence detection and identification of cracks in dents. This derived in a performance specification stating the truth capabilities to detect interacting features in a dent. These learnings were applied to commercial inspections where the feedback loop is closed with the field verifications.

Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, the long (ZAP-L) and short (ZAP-S), is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP decelerates the progression of HCMV infection. SLAM-sequencing revealed that ZAP restricts HCMV at early stages of infection by destabilising a distinct subset of viral transcripts with low CG content. In summary, this report provides evidence of an important antiviral role for ZAP in host defense against HCMV infection and highlights its differentiated function during DNA virus infection.

Herpes simplex virus 1 (HSV-1) installs a profound host shut-off during lytic infection. The virion host shut-off (vhs) protein plays a key role in this process by efficiently cleaving both host and viral mRNAs in a translation-initiation-dependent manner. Furthermore, the onset of viral DNA replication is accompanied by a rapid decline in transcriptional activity of the host genome. Both mechanisms have tremendous impact on the RNA expression profile of the infected cells. To dissect their relative contributions and elucidate gene-specific host transcriptional responses throughout the first 8h of lytic HSV-1 infection, we here employed RNA-seq of total, newly transcribed (4sU-labelled) and chromatin-associated RNA in wild-type (WT) and Δvhs infection of primary human fibroblasts. Following virus entry, vhs activity rapidly plateaued at an elimination rate of around 30% of cellular mRNAs per hour until 8h p.i. In parallel, host transcriptional activity dropped down to 10-20%. While the combined effects of both phenomena dominated infection-induced changes in total RNA, extensive gene-specific transcriptional regulation was observable in chromatin-associated RNA. This was surprisingly concordant between WT HSV-1 and its Δvhs mutant and at least in parts mediated by the embryonic transcription factor DUX4. Furthermore, both WT and Δvhs infection induced strong transcriptional up-regulation of a small subset of genes. Most of these were either poorly or not at all expressed prior to infection but already primed by H3K4me3 histone marks at their promoters. Most interestingly, analysis of chromatin-associated RNA revealed vhs-nuclease-activity-dependent transcriptional down-regulation of at least 150 cellular genes, in particular of many genes encoding integrin adhesome and extracellular matrix components. This was accompanied by a vhs-dependent reduction in protein levels by 8h p.i. for many of these genes. In summary, our study provides a comprehensive picture of the molecular mechanisms that govern cellular RNA metabolism during the first 8h of lytic HSV-1 infection.Author SummaryThe HSV-1 virion host shut-off (vhs) protein efficiently cleaves both host and viral mRNAs in a translation-dependent manner. In this study, we model and quantify changes in vhs activity as well as the virus-induced global loss of host transcriptional activity during productive HSV-1 infection. In general, HSV-1-induced alterations in total RNA levels were found to be predominantly shaped by these two global processes rather than gene-specific regulation. In contrast, chromatin-associated RNA depicted gene-specific transcriptional changes. This revealed highly concordant transcriptional changes in WT and Δvhs infection, confirmed DUX4 as a key transcriptional regulator in HSV-1 infection and depicted vhs-dependent, transcriptional down-regulation of the integrin adhesome and extracellular matrix. The latter explained some of the gene-specific effects previously attributed to vhs-mediated mRNA degradation and resulted in a concordant loss in protein levels by 8h p.i. for many of the respective genes.

Small hydropower (SHP) has a global reputation as a ‘green’, low-carbon energy source that improves rural livelihoods and contributes to local economic development. In China, SHP has grown rapidly since the early 2000s, particularly in the water-rich provinces in the country’s southwest. However, because SHP plants in China are privately-operated and approved by local governments, there is an incentive to construct large, multiple-cascade systems to generate as much power as possible. In many areas, this has led to over-development of SHP and associated negative environmental and social impacts. In this paper, we identify the factors that shape geographies of SHP over-development, what we refer to as ‘shades’ of green energy. We then analyze the direct and indirect impacts of over-development. We draw on interviews and electricity generation data from six prefectures in Yunnan province, one of the world’s largest hydropower producing regions. We find that prefectures that operate in a semi-autonomous way in electricity management and industrial planning are most prone to over-develop SHP, since they depend on hydropower revenues from electricity generation and local energy-intensive industries. We also find that over-development of SHP causes streamflow reductions and unstable electricity generation, and in some areas, drives an increase in environmentally-destructive mineral processing and reduces irrigation water access. These findings suggest a need for coordinated river basin planning on small watersheds and a reassessment of the role of SHP in local economic development.

SummarySince the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.

DNA damage response (DDR) involves dramatic transcriptional alterations, the mechanisms of which remain ill defined. Here, we show that following genotoxic stress, the RNA-binding motif protein 7 (RBM7) stimulates RNA polymerase II (Pol II) transcription and promotes cell viability by activating the positive transcription elongation factor b (P-TEFb) via its release from the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP). This is mediated by activation of p38MAPK, which triggers enhanced binding of RBM7 with core subunits of 7SK snRNP. In turn, P-TEFb relocates to chromatin to induce transcription of short units, including key DDR genes and multiple classes of non-coding RNAs. Critically, interfering with the axis of RBM7 and P-TEFb provokes cellular hypersensitivity to DNA-damage-inducing agents due to activation of apoptosis. Our work uncovers the importance of stress-dependent stimulation of Pol II pause release, which enables a pro-survival transcriptional response that is crucial for cell fate upon genotoxic insult.

The Ayeyarwady basin, one of Asia's largest transnational river basins, shared by Myanmar, China and India, has an immense theoretical hydropower potential of 45 GW. Currently hydropower projects with an approximate capacity of 33 GW are already in various stages of development. Projects include some of the world's largest and most controversial endeavours (e.g. Myitsone). Of these projects 6.4 GW are already installed in 187 hydropower projects (≥1 MW). Despite its huge relevance, the transnational Ayeyarwady basin still belongs to the less known basins, which is largely attributed to its small Chinese section. However, the current knowledge is limited to Myanmar's segment where a limited number of 16 projects exist. In contrast, there are a multitude of projects −174 (18 large and 156 small) - in the Chinese section, including the basins largest one (Daying-4: 875 MW). The combination of small and large projects makes the Chinese section one of the most dense (small) hydropower catchments worldwide. About half of the entire basin's hydropower output is exported to the energy starved Guangdong province (from both countries) and almost another quarter is used for energy intensive industries in the tiny Chinese section; hence currently only about 30% of the basin's generated (hydro-)electricity is used there for households, industry, etc. This results in a complex water-energy nexus that is complicated by a tangled geopolitical setting directly in the border region, where many projects are located. This paper describes that nexus based on Darrin Magee's powershed approach.

Since the late 1980’s Ultrasonic tools have been used for the detection and sizing of crack like indications. ILI service providers developed inspection technologies for liquid and gas lines that are widely used nowadays. In comparison to axial cracking, circumferential cracking is not a prevalent risk to most pipelines and therefore is not as well understood. Nevertheless, pipeline Operators observe from time to time circumferentially oriented defects, often in combination with circumferential welds or local stress/strain accumulations. These are often caused by pipeline movement, which may especially occur in mountain areas. With the introduction of Ultrasonic circumferential crack inspection tools in the late 2000’s the knowledge has steadily increased over time. Extensive data collected from in-ditch NDE validations has provided NDT Global with an increased knowledge of the morphology of single cracking and stress corrosion cracking defects both in the axial and circumferential orientations. Field verifications have shown that not all features have the same morphology. Some of the challenges with circumferential cracking are for features that fall outside of the industry standard specifications. These types of features can exhibit characteristics such as being sloped, skewed or tilted. In 2016 NDT Global was approached by Plains Midstream Canada to complete inspections utilizing the 10″ Ultrasonic Circumferential crack inspection technology. The pipeline system spans 188km within Canada and consists of 2 segments. The pipeline traverses several elevation changes and crosses several creeks and roads. Circumferential cracking was identified during dig campaigns performed for other threats, therefore the need to inspect each pipeline segment with the Ultrasonic circumferential technology was identified. Plains Midstream Canada and NDT Global formed a close collaboration to assess the severity of circumferential crack features in this line. This paper will discuss integrity aspects from an Operator and Vendor perspective. Challenges identified due to the morphology of the circumferential crack like indications and derived analysis rules and interpretation methodologies to optimize characterization and sizing are presented. Finally, potential opportunities to maintain the integrity of similar assets by applying some of the findings and enhance the management and decision making process are suggested.