Gwenny Cackett

Nucleocytoplasmic Large DNA Viruses (NCLDV) including African swine fever virus (ASFV) and poxviruses encode an understudied viral RNA polymerase (vRNAP) transcription system related to archaeal RNAP and eukaryotic RNA polymerase II. Immediately upon infection, virus-specific transcription initiation factors, which are included in the virus particle, enable early gene transcription even before virions have fully disassembled. We have produced a recombinant form of ASFV Early Transcription Factor (AETF) in insect cells, and show that it is recruited to promoters dependent on the Early Promoter Motif (EPM). The 3.9 Å resolution cryo-EM structure of the 284 kDa heterodimer reveals a complex domain composition and topology. The large AETF1 subunit contains two DNA-binding domains: a host-related TATA-binding protein-like domain and a virus-specific EPM-recognition domain, as well as an embedded ubiquitin-like domain, and a pseudo-deubiquitinase domain. We found that recombinant AETF1 is ubiquitinylated, indicative of early gene regulation by small protein modifiers. The smaller AETF2 subunit includes a canonical bi-lobal helicase domain with DNA-independent ATPase activity, likely involved in DNA melting and template loading analogous to TFIIH. Systematic sequence and structure comparisons evidence that AETF is a prototypical ancestral early factor in NCLDVs.

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.

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.

African Swine Fever Virus (ASFV) is a Nucleo-Cytoplasmic Large DNA Virus which causes an incurable haemorrhagic fever in wild and domesticated 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. Little is known about how this system works at a molecular level and there are currently no approved vaccines nor antiviral drugs to treat the disease. Here, we present methods to produce recombinant ASFV RNA polymerase (RNAP), functional assays suitable to screen for inhibitors, and high-resolution cryo-Electron Microscopy structures of the viral RNAP in different conformational states. The viral RNAP bears a striking resemblance to RNAPII with bona fide homologs of nine of its twelve subunits. Key differences include the fusion of 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-methyl transferase 1. Despite the structural conservation with eukaryotic and bacterial polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin, offering the grounds for the development of highly selective inhibitors. 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.

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

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.

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 hpi. 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 newly characterised MGF 100 genes I7L and I8L. Structural homology modelling suggest that I7L and I8L encode unorthodox SH2 domain proteins with the potential to interfere with the host’s immune response. In parallel, we monitored transcriptome changes in the infected host cells, which showed a pro-inflammatory immune response with the upregulation of NF-kB activated genes, innate immunity, as well as lysosome components including S100 proteins. Author Summary African swine fever virus (ASFV) causes a haemorrhagic fever in domestic pigs and wild boar with mortality rates approaching 100%, for which there are no approved vaccines or antivirals. The highly-virulent ASFV Georgia 2007/1 strain was the first isolated when ASFV spread from Africa to the Caucasus region in 2007. From here it has spread through Eastern Europe, and more recently across Asia. We have used an RNA-based next generation sequencing technique called CAGE-seq to map the starts of viral genes across the ASFV Georgia 2007/1 strain DNA genome. This has allowed us to investigate how it controls its viral gene expression during different stages of infection in macrophage cells. We have characterised which genes are expressed at different levels during early or late stages of infection, and compared them to the non-virulent ASFV-BA71V strain to identify key genes that enhance virulence. We have discovered new genes, and predicted the likely roles of uncharacterised genes during ASFV infection. In parallel we have investigated how the host cells respond to ASFV infection, which has revealed how the virus early on suppresses components of the host immune response to ultimately win the arms race against its porcine host.

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.

The African Swine Fever Virus causes haemorrhagic fever in domestic pigs and presents the biggest global threat to animal farming in recorded history. Despite its importance, very little is known the mechanisms and temporal regulation of transcription in ASFV. Here we report the first detailed viral transcriptome analysis of ASFV during early and late infection ofVerocells. In addition to total RNA sequencing, we have characterised the transcription start sites and transcription termination sites at nucleotide-resolution, revealing the distinct DNA consensus motifs of early and late promoters, as well as the sequence determinants for transcription termination. ASFV can utilise alternative promoters to generate distinct proteins from the same transcription unit that differ with respect to the polypeptide N-terminus. Finally, our results reveal that the ASFV-RNAP undergoes transcript slippage at the 5’ end of transcription units that in a promoter sequence-specific manner results in the addition of 5’-AT and 5’-ATAT tails to mRNAs.