Helicases DDX5 and DDX17 promote heterogeneity in HBV transcription termination in infected human hepatocytes.

BACKGROUND & AIMS: Transcription termination fine tunes gene expression and contributes to specify the function of RNAs in eukaryotic cells. Transcription termination of hepatitis B virus (HBV) is subjected to the recognition of the canonical polyadenylation signal (cPAS) common to all viral transcripts. The regulation of the usage of this cPAS and its impact on viral gene expression and replication is currently unknown. APPROACH & RESULTS: To unravel the regulation of HBV transcript termination, we implemented a 3' RACE-PCR assay coupled to single molecule sequencing both in in vitro infected hepatocytes and in chronically infected patients. The detection of a previously unidentified transcriptional readthrough indicated that the cPAS was not systematically recognized during HBV replication in vitro and in vivo. Gene expression downregulation experiments demonstrated a role for the RNA helicases DDX5 and DDX17 in promoting viral transcriptional readthrough, which was, in turn, associated to HBV RNA destabilization and decreased HBx protein expression. RNA and chromatin immunoprecipitation, together with mutation of cPAS sequence, suggested a direct role of DDX5 and DDX17 in functionally linking cPAS recognition to transcriptional readthrough, HBV RNA stability and replication. CONCLUSIONS: Our findings identify DDX5 and DDX17 as crucial determinants for HBV transcriptional fidelity and as host restriction factors for HBV replication. IMPACT AND IMPLICATIONS: Hepatitis B virus (HBV) covalently closed circular (ccc)DNA degradation or functional inactivation remains the holy grail to be attained to achieve HBV cure. Transcriptional fidelity is a cornerstone in gene expression regulation. Here, we demonstrate that two helicases, DDX5 and DDX17, inhibit the recognition of HBV polyadenylation signal and transcriptional termination, thus decreasing HBV RNA stability and acting as restriction factors for efficient cccDNA transcription and viral replication. The observation that DDX5 and DDX17 are downregulated in HBV chronically infected patients suggests a role for the helicases in HBV persistence in vivo. These results open new perspectives for researchers aiming at identifying new targets to neutralise cccDNA transcription.

TLR8 agonist selgantolimod regulates Kupffer cell differentiation status and impairs HBV entry into hepatocytes via an IL-6-dependent mechanism.

OBJECTIVE: Achieving HBV cure will require novel combination therapies of direct-acting antivirals and immunomodulatory agents. In this context, the toll-like receptor 8 (TLR8) agonist selgantolimod (SLGN) has been investigated in preclinical models and clinical trials for chronic hepatitis B (CHB). However, little is known regarding its action on immune effectors within the liver. Our aim was to characterise the transcriptomic changes and intercellular communication events induced by SLGN in the hepatic microenvironment. DESIGN: We identified TLR8-expressing cell types in the human liver using publicly available single-cell RNA-seq data and established a method to isolate Kupffer cells (KCs). We characterised transcriptomic and cytokine KC profiles in response to SLGN. SLGN's indirect effect was evaluated by RNA-seq in hepatocytes treated with SLGN-conditioned media (CM) and quantification of HBV parameters following infection. Pathways mediating SLGN's effect were validated using transcriptomic data from HBV-infected patients. RESULTS: Hepatic TLR8 expression takes place in the myeloid compartment. SLGN treatment of KCs upregulated monocyte markers (eg, S100A12) and downregulated genes associated with the KC identity (eg, SPIC). Treatment of hepatocytes with SLGN-CM downregulated NTCP and impaired HBV entry. Cotreatment with an interleukin 6-neutralising antibody reverted the HBV entry inhibition. CONCLUSION: Our transcriptomic characterisation of SLGN sheds light into the programmes regulating KC activation. Furthermore, in addition to its previously described effect on established HBV infection and adaptive immunity, we show that SLGN impairs HBV entry. Altogether, SLGN may contribute through KCs to remodelling the intrahepatic immune microenvironment and may thus represent an important component of future combinations to cure HBV infection.

Interspecies comparison of the early transcriptomic changes associated with hepatitis B virus exposure in human and macaque immune cell populations.

Background and aims: Hepatitis B virus (HBV) infection affects 300 million individuals worldwide, representing a major factor for the development of hepatic complications. Although existing antivirals are effective in suppressing replication, eradication of HBV is not achieved. Therefore, a multi-faceted approach involving antivirals and immunomodulatory agents is required. Non-human primates are widely used in pre-clinical studies due to their close evolutionary relationship to humans. Nonetheless, it is fundamental to identify the differences in immune response between humans and these models. Thus, we performed a transcriptomic characterization and interspecies comparison of the early immune responses to HBV in human and cynomolgus macaques. Methods: We characterized early transcriptomic changes in human and cynomolgus B cells, T cells, myeloid and plasmacytoid dendritic cells (pDC) exposed to HBV ex vivo for 2 hours. Differentially-expressed genes were further compared to the profiles of HBV-infected patients using publicly-available single-cell data. Results: HBV induced a wide variety of transcriptional changes in all cell types, with common genes between species representing only a small proportion. In particular, interferon gamma signaling was repressed in human pDCs. At the gene level, interferon gamma inducible protein 16 (IFI16) was upregulated in macaque pDCs, while downregulated in humans. Moreover, IFI16 expression in pDCs from chronic HBV-infected patients anti-paralleled serum HBsAg levels. Conclusion: Our characterization of early transcriptomic changes induced by HBV in humans and cynomolgus macaques represents a useful resource for the identification of shared and divergent host responses, as well as potential immune targets against HBV.

An SSR-SNP Linkage Map of the Parasitic Weed Orobanche cumana Wallr. Including a Gene for Plant Pigmentation.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant that causes major yield losses to sunflower crops in the Old World. Efforts to understand how this parasitic weed recognizes and interacts with sunflowers are important for developing long-term genetic resistance strategies. However, such studies are hampered by the lack of genetic tools for O. cumana. The objectives of this research were to construct a genetic linkage map of this species using SSR and SNP markers, and mapping the Pg locus that is involved in plant pigmentation. The genetic map was developed from the progenies of a cross between the O. cumana inbred lines EK-12 and EK-A1, which originated from populations belonging to two distant and geographically separated gene pools identified in Spain. The inbred lines also differed in plant pigmentation, with EK-A1 lacking anthocyanin pigmentation (pgpg genotype). A genetic map comprising 26 SSR and 701 SNP markers was constructed, which displayed 19 linkage groups (LGs), corresponding to the 19 chromosome pairs of O. cumana. The total length of the map was 1795.7 cM, with an average distance between two adjacent positions of 2.5 cM and a maximum map distance of 41.9 cM. The Pg locus mapped to LG19 between the SNP markers OS02468 and OS01653 at 7.5 and 3.4 cM, respectively. This study constitutes the first linkage map and trait mapping study in Orobanche spp., laying a key foundation for further genome characterization and providing a basis for mapping additional traits such as those having a key role in parasitism.

Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread.

Plant immune receptors of the class of nucleotide-binding and leucine-rich repeat domain (NLR) proteins can contain additional domains besides canonical NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4 (NB-ARC)) and leucine-rich repeat (LRR) domains. Recent research suggests that these additional domains act as integrated decoys recognizing effectors from pathogens. Proteins homologous to integrated decoys are suspected to be effector targets and involved in disease or resistance. Here, we scrutinized 31 entire plant genomes to identify putative integrated decoy domains in NLR proteins using the Interpro search. The involvement of the Zinc Finger-BED type (ZBED) protein containing a putative decoy domain, called BED, in rice (Oryza sativa) resistance was investigated by evaluating susceptibility to the blast fungus Magnaporthe oryzae in rice over-expression and knock-out mutants. This analysis showed that all plants tested had integrated various atypical protein domains into their NLR proteins (on average 3.5% of all NLR proteins). We also demonstrated that modifying the expression of the ZBED gene modified disease susceptibility. This study suggests that integration of decoy domains in NLR immune receptors is widespread and frequent in plants. The integrated decoy model is therefore a powerful concept to identify new proteins involved in disease resistance. Further in-depth examination of additional domains in NLR proteins promises to unravel many new proteins of the plant immune system.

Identification of positive and negative regulators of disease resistance to rice blast fungus using constitutive gene expression patterns.

Elevated constitutive expression of components of the defence arsenal is associated with quantitative resistance to the rice blast fungus, a phenomenon called preformed defence. While the role of many disease regulators in inducible defence systems has been extensively studied, little attention has been paid so far to genes that regulate preformed defence. In this study, we show by microarray analysis across rice diversity that the preformed defence phenomenon impacts on a large number of defence-related genes without apparently affecting other biological processes. Using a guilt-by-association strategy, we identified two positive regulators that promote constitutive expression of known defence markers and partial resistance to rice blast. The HSF23 gene encodes for a putative member of the heat shock transcription factor family, while CaMBP encodes for a putative Calmodulin-binding protein. Both HSF23 and CaMBP strongly affect preformed defence and also plant growth. Additionally, we identified the OB-fold gene as a negative regulator of blast resistance, which could be involved in RNA stabilization. The OB-fold mutants do not suffer from obvious developmental defects. Taken together, our results prove that our strategy of combining analysis of gene expression diversity with guilt-by-association is a powerful way to identify disease resistance regulators in rice.

Preformed expression of defense is a hallmark of partial resistance to rice blast fungal pathogen Magnaporthe oryzae.

BACKGROUND: Partial resistance to plant pathogens is extensively used in breeding programs since it could contribute to resistance durability. Partial resistance often builds up during plant development and confers quantitative and usually broad-spectrum resistance. However, very little is known on the mechanisms underlying partial resistance. Partial resistance is often explained by poorly effective induction of plant defense systems. By exploring rice natural diversity, we asked whether expression of defense systems before infection could explain partial resistance towards the major fungal pathogen Magnaporthe oryzae. The constitutive expression of 21 defense-related genes belonging to the defense system was monitored in 23 randomly sampled rice cultivars for which partial resistance was measured. RESULTS: We identified a strong correlation between the expression of defense-related genes before infection and partial resistance. Only a weak correlation was found between the induction of defense genes and partial resistance. Increasing constitutive expression of defense-related genes also correlated with the establishment of partial resistance during plant development. Some rice genetic sub-groups displayed a particular pattern of constitutive expression, suggesting a strong natural polymorphism for constitutive expression of defense. Constitutive levels of hormones like salicylic acid and ethylene cannot explain constitutive expression of defense. We could identify an area of the genome that contributes to explain both preformed defense and partial resistance. CONCLUSION: These results indicate that constitutive expression of defense-related genes is likely responsible for a large part of partial resistance in rice. The finding of this preformed defense system should help guide future breeding programs and open the possibility to identify the molecular mechanisms behind partial resistance.