Transcription termination and readthrough in African swine fever virus.

Introduction: African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that encodes its own host-like RNA polymerase (RNAP) and factors required to produce mature mRNA. The formation of accurate mRNA 3' ends by ASFV RNAP depends on transcription termination, likely enabled by a combination of sequence motifs and transcription factors, although these are poorly understood. The termination of any RNAP is rarely 100% efficient, and the transcriptional "readthrough" at terminators can generate long mRNAs which may interfere with the expression of downstream genes. ASFV transcriptome analyses reveal a landscape of heterogeneous mRNA 3' termini, likely a combination of bona fide termination sites and the result of mRNA degradation and processing. While short-read sequencing (SRS) like 3' RNA-seq indicates an accumulation of mRNA 3' ends at specific sites, it cannot inform about which promoters and transcription start sites (TSSs) directed their synthesis, i.e., information about the complete and unprocessed mRNAs at nucleotide resolution. Methods: Here, we report a rigorous analysis of full-length ASFV transcripts using long-read sequencing (LRS). We systematically compared transcription termination sites predicted from SRS 3' RNA-seq with 3' ends mapped by LRS during early and late infection. Results: Using in-vitro transcription assays, we show that recombinant ASFV RNAP terminates transcription at polyT stretches in the non-template strand, similar to the archaeal RNAP or eukaryotic RNAPIII, unaided by secondary RNA structures or predicted viral termination factors. Our results cement this T-rich motif (U-rich in the RNA) as a universal transcription termination signal in ASFV. Many genes share the usage of the same terminators, while genes can also use a range of terminators to generate transcript isoforms varying enormously in length. A key factor in the latter phenomenon is the highly abundant terminator readthrough we observed, which is more prevalent during late compared with early infection. Discussion: This indicates that ASFV mRNAs under the control of late gene promoters utilize different termination mechanisms and factors to early promoters and/or that cellular factors influence the viral transcriptome landscape differently during the late stages of infection.

Structure of the recombinant RNA polymerase from African Swine Fever Virus.

African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.

Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays.

CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together 'chimeric' chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays.

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface.

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

A novel metagenome-derived viral RNA polymerase and its application in a cell-free expression system for metagenome screening.

The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, EM1 RNAP, identified from a metagenome data set obtained from an elephant dung microbiome. EM1 RNAP and its promoter sequence are distantly related to T7 RNA polymerase. Using EM1 RNAP and a translation-competent Escherichia coli extract, we have developed an efficient medium-throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method.

A prebiotic basis for ATP as the universal energy currency.

ATP is universally conserved as the principal energy currency in cells, driving metabolism through phosphorylation and condensation reactions. Such deep conservation suggests that ATP arose at an early stage of biochemical evolution. Yet purine synthesis requires 6 phosphorylation steps linked to ATP hydrolysis. This autocatalytic requirement for ATP to synthesize ATP implies the need for an earlier prebiotic ATP equivalent, which could drive protometabolism before purine synthesis. Why this early phosphorylating agent was replaced, and specifically with ATP rather than other nucleoside triphosphates, remains a mystery. Here, we show that the deep conservation of ATP might reflect its prebiotic chemistry in relation to another universally conserved intermediate, acetyl phosphate (AcP), which bridges between thioester and phosphate metabolism by linking acetyl CoA to the substrate-level phosphorylation of ADP. We confirm earlier results showing that AcP can phosphorylate ADP to ATP at nearly 20% yield in water in the presence of Fe3+ ions. We then show that Fe3+ and AcP are surprisingly favoured. A wide range of prebiotically relevant ions and minerals failed to catalyse ADP phosphorylation. From a panel of prebiotic phosphorylating agents, only AcP, and to a lesser extent carbamoyl phosphate, showed any significant phosphorylating potential. Critically, AcP did not phosphorylate any other nucleoside diphosphate. We use these data, reaction kinetics, and molecular dynamic simulations to infer a possible mechanism. Our findings might suggest that the reason ATP is universally conserved across life is that its formation is chemically favoured in aqueous solution under mild prebiotic conditions.

Proteome Analysis of Swine Macrophages after Infection with Two Genotype II African Swine Fever Isolates of Different Pathogenicity.

Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.

How to Shut Down Transcription in Archaea during Virus Infection.

Multisubunit RNA polymerases (RNAPs) carry out transcription in all domains of life; during virus infection, RNAPs are targeted by transcription factors encoded by either the cell or the virus, resulting in the global repression of transcription with distinct outcomes for different host-virus combinations. These repressors serve as versatile molecular probes to study RNAP mechanisms, as well as aid the exploration of druggable sites for the development of new antibiotics. Here, we review the mechanisms and structural basis of RNAP inhibition by the viral repressor RIP and the crenarchaeal negative regulator TFS4, which follow distinct strategies. RIP operates by occluding the DNA-binding channel and mimicking the initiation factor TFB/TFIIB. RIP binds tightly to the clamp and locks it into one fixed position, thereby preventing conformational oscillations that are critical for RNAP function as it progresses through the transcription cycle. TFS4 engages with RNAP in a similar manner to transcript cleavage factors such as TFS/TFIIS through the NTP-entry channel; TFS4 interferes with the trigger loop and bridge helix within the active site by occlusion and allosteric mechanisms, respectively. The conformational changes in RNAP described above are universally conserved and are also seen in inactive dimers of eukaryotic RNAPI and several inhibited RNAP complexes of both bacterial and eukaryotic RNA polymerases, including inactive states that precede transcription termination. A comparison of target sites and inhibitory mechanisms reveals that proteinaceous repressors and RNAP-specific antibiotics use surprisingly common ways to inhibit RNAP function.

Progress and Challenges in Archaeal Molecular Biology.

Archaea are a key feature of the terran biosphere. Since their early characterization in the 1970s, we have learned much about the fascinating organism, most recently by applying genome-, transcriptome-, proteome-, and metabolome-scale methods. This chapter describes seminal contributions that elaborate on the study of archaeal biology at the systems level.

ChIP-Seq Occupancy Mapping of the Archaeal Transcription Machinery.

Genome-wide occupancy studies for RNA polymerases and their basal transcription factors deliver information about transcription dynamics and the recruitment of transcription elongation and termination factors in eukaryotes and prokaryotes. The primary method to determine genome-wide occupancies is chromatin immunoprecipitation combined with deep sequencing (ChIP-seq). Archaea possess a transcription machinery that is evolutionarily closer related to its eukaryotic counterpart but it operates in a prokaryotic cellular context. Studies on archaeal transcription brought insight into the evolution of transcription machineries and the universality of transcription mechanisms. Because of the limited resolution of ChIP-seq, the close spacing of promoters and transcription units found in archaeal genomes pose a challenge for ChIP-seq and the ensuing data analysis. The extreme growth temperature of many established archaeal model organisms necessitates further adaptations. This chapter describes a version of ChIP-seq adapted for the basal transcription machinery of thermophilic archaea and some modifications to the data analysis.

African Swine Fever Virus and Host Response: Transcriptome Profiling of the Georgia 2007/1 Strain and Porcine Macrophages.

African swine fever virus (ASFV) has a major global economic impact. With a case fatality in domestic pigs approaching 100%, it currently presents the largest threat to animal farming. Although genomic differences between attenuated and highly virulent ASFV strains have been identified, the molecular determinants for virulence at the level of gene expression have remained opaque. Here, we characterize the transcriptome of ASFV genotype II Georgia 2007/1 (GRG) during infection of the physiologically relevant host cells, porcine macrophages. In this study, we applied cap analysis gene expression sequencing (CAGE-seq) to map th0e 5' ends of viral mRNAs at 5 and 16 h postinfection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterize the global early and late promoter landscape of GRG. We compared transcriptome maps of the GRG isolate and the lab-attenuated BA71V strain that highlighted GRG virulence-specific transcripts belonging to multigene families, including two predicted MGF 100 genes, I7L and I8L. In parallel, we monitored transcriptome changes in the infected host macrophage cells. Of the 9,384 macrophage genes studied, transcripts for 652 host genes were differentially regulated between 5 and 16 h postinfection compared with only 25 between uninfected cells and 5 h postinfection. NF-κB activated genes and lysosome components such as S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5, and CXCL8 were downregulated. IMPORTANCE African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, with case fatality rates approaching 100% and no approved vaccines or antivirals. The highly virulent ASFV Georgia 2007/1 strain (GRG) was the first isolated when ASFV spread from Africa to the Caucasus region in 2007, then spreading through Eastern Europe and, more recently, across Asia. We used an RNA-based next-generation sequencing technique called CAGE-seq to map the starts of viral genes across the GRG DNA genome. This has allowed us to investigate which viral genes are expressed during early or late stages of infection and how this is controlled, comparing their expression to the nonvirulent ASFV-BA71V strain to identify key genes that play a role in virulence. In parallel, we investigated how host cells respond to infection, which revealed how the ASFV suppresses components of the host immune response to ultimately win the arms race against its porcine host.

Structural basis of RNA polymerase inhibition by viral and host factors.

RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.

Promoter-proximal elongation regulates transcription in archaea.

Recruitment of RNA polymerase and initiation factors to the promoter is the only known target for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation are involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Saccharolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.

Coupling of Transcription and Translation in Archaea: Cues From the Bacterial World.

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae. Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea.

Comparative CRISPR type III-based knockdown of essential genes in hyperthermophilic Sulfolobales and the evasion of lethal gene silencing.

CRISPR type III systems, which are abundantly found in archaea, recognize and degrade RNA in their specific response to invading nucleic acids. Therefore, these systems can be harnessed for gene knockdown technologies even in hyperthermophilic archaea to study essential genes. We show here the broader usability of this posttranscriptional silencing technology by expanding the application to further essential genes and systematically analysing and comparing silencing thresholds and escape mutants. Synthetic guide RNAs expressed from miniCRISPR cassettes were used to silence genes involved in cell division (cdvA), transcription (rpo8), and RNA metabolism (smAP2) of the two crenarchaeal model organisms Saccharolobus solfataricus and Sulfolobus acidocaldarius. Results were systematically analysed together with those obtained from earlier experiments of cell wall biogenesis (slaB) and translation (aif5A). Comparison of over 100 individual transformants revealed gene-specific silencing maxima ranging between 40 and 75%, which induced specific knockdown phenotypes leading to growth retardation. Exceedance of this threshold by strong miniCRISPR constructs was not tolerated and led to specific mutation of the silencing miniCRISPR array and phenotypical reversion of cultures. In two thirds of sequenced reverted cultures, the targeting spacers were found to be precisely excised from the miniCRISPR array, indicating a still hypothetical, but highly active recombination system acting on the dynamics of CRISPR spacer arrays. Our results indicate that CRISPR type III - based silencing is a broadly applicable tool to study in vivo functions of essential genes in Sulfolobales which underlies a specific mechanism to avoid malignant silencing overdose.

Transcriptome view of a killer: African swine fever virus.

African swine fever virus (ASFV) represents a severe threat to global agriculture with the world's domestic pig population reduced by a quarter following recent outbreaks in Europe and Asia. Like other nucleocytoplasmic large DNA viruses, ASFV encodes a transcription apparatus including a eukaryote-like RNA polymerase along with a combination of virus-specific, and host-related transcription factors homologous to the TATA-binding protein (TBP) and TFIIB. Despite its high impact, the molecular basis and temporal regulation of ASFV transcription is not well understood. Our lab recently applied deep sequencing approaches to characterise the viral transcriptome and gene expression during early and late ASFV infection. We have characterised the viral promoter elements and termination signatures, by mapping the RNA-5' and RNA-3' termini at single nucleotide resolution. In this review, we discuss the emerging field of ASFV transcripts, transcription, and transcriptomics.

The African Swine Fever Virus Transcriptome.

African swine fever virus (ASFV) causes hemorrhagic fever in domestic pigs, presenting the biggest global threat to animal farming in recorded history. Despite the importance of ASFV, little is known about the mechanisms and regulation of ASFV transcription. Using RNA sequencing methods, we have determined total RNA abundance, transcription start sites, and transcription termination sites at single-nucleotide resolution. This allowed us to characterize DNA consensus motifs of early and late ASFV core promoters, as well as a polythymidylate sequence determinant for transcription termination. Our results demonstrate that ASFV utilizes alternative transcription start sites between early and late stages of infection and that ASFV RNA polymerase (RNAP) undergoes promoter-proximal transcript slippage at 5' ends of transcription units, adding quasitemplated AU- and AUAU-5' extensions to mRNAs. Here, we present the first much-needed genome-wide transcriptome study that provides unique insight into ASFV transcription and serves as a resource to aid future functional analyses of ASFV genes which are essential to combat this devastating disease.IMPORTANCE African swine fever virus (ASFV) causes incurable and often lethal hemorrhagic fever in domestic pigs. In 2020, ASF presents an acute and global animal health emergency that has the potential to devastate entire national economies as effective vaccines or antiviral drugs are not currently available (according to the Food and Agriculture Organization of the United Nations). With major outbreaks ongoing in Eastern Europe and Asia, urgent action is needed to advance our knowledge about the fundamental biology of ASFV, including the mechanisms and temporal control of gene expression. A thorough understanding of RNAP and transcription factor function, and of the sequence context of their promoter motifs, as well as accurate knowledge of which genes are expressed when and the amino acid sequence of the encoded proteins, is direly needed for the development of antiviral drugs and vaccines.

Key Concepts and Challenges in Archaeal Transcription.

Transcription is enabled by RNA polymerase and general factors that allow its progress through the transcription cycle by facilitating initiation, elongation and termination. The transitions between specific stages of the transcription cycle provide opportunities for the global and gene-specific regulation of gene expression. The exact mechanisms and the extent to which the different steps of transcription are exploited for regulation vary between the domains of life, individual species and transcription units. However, a surprising degree of conservation is apparent. Similar key steps in the transcription cycle can be targeted by homologous or unrelated factors providing insights into the mechanisms of RNAP and the evolution of the transcription machinery. Archaea are bona fide prokaryotes but employ a eukaryote-like transcription system to express the information of bacteria-like genomes. Thus, archaea provide the means not only to study transcription mechanisms of interesting model systems but also to test key concepts of regulation in this arena. In this review, we discuss key principles of archaeal transcription, new questions that still await experimental investigation, and how novel integrative approaches hold great promise to fill this gap in our knowledge.

Structural and functional adaptation of Haloferax volcanii TFEα/ß.

The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEß subunit. Halophilic archaea such as Haloferax volcanii have highly divergent putative TFEß homologues with unknown properties. Here, we demonstrate that Haloferax TFEß lacks the prototypical iron-sulphur cluster yet still forms a stable complex with TFEα. A second metal cluster contained in the zinc ribbon domain in TFEα is highly degenerate but retains low binding affinity for zinc, which contributes to protein folding and stability. The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Our results reveal the loss of metal centres in Hvo transcription factors, and confirm the dual function of TFE as basal factor and regulator of transcription.