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.

This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemicalmapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg .fr/rnapuzzles/.

PIN-like domains constitute a widespread superfamily of nucleases, diverse in terms of the reaction mechanism, substrate specificity, biological function and taxonomic distribution. Proteins with PIN-like domains are involved in central cellular processes, such as DNA replication and repair, mRNA degradation, transcription regulation and ncRNA maturation. In this work, we identify and classify the most complete set of PIN-like domains to provide the first comprehensive analysis of sequence–structure–function relationships within the whole PIN domain-like superfamily. Transitive sequence searches using highly sensitive methods for remote homology detection led to the identification of several new families, including representatives of Pfam (DUF1308, DUF4935) and CDD (COG2454), and 23 other families not classified in the public domain databases. Further sequence clustering revealed relationships between individual sequence clusters and showed heterogeneity within some families, suggesting a possible functional divergence. With five structural groups, 70 defined clusters, over 100,000 proteins, and broad biological functions, the PIN domain-like superfamily constitutes one of the largest and most diverse nuclease superfamilies. Detailed analyses of sequences and structures, domain architectures, and genomic contexts allowed us to predict biological function of several new families, including new toxin-antitoxin components, proteins involved in tRNA/rRNA maturation and transcription/translation regulation.

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 characterise 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 the 5’ ends of viral mRNAs at 5 and 16 hours post-infection. A bioinformatics analysis of the sequence context surrounding the transcription start sites (TSSs) enabled us to characterise 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 virulent-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 hours-post-infection compared with only 25 between uninfected cells and 5 hours post-infection. NF-kB activated genes and lysosome components like S100 were upregulated, and chemokines such as CCL24, CXCL2, CXCL5 and CXCL8 downregulated. African swine fever virus (ASFV) causes haemorrhagic 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 non-virulent 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.