RNR-R2 Upregulation by a Short Non-Coding Viral Transcript

DNA viruses require dNTPs for replication and have developed different strategies to increase intracellular dNTP pools. Hepatitis B virus (HBV) infects non-dividing cells in which dNTPs are scarce and the question is how viral replication takes place. Previously we reported that the virus induces the DNA damage response (DDR) pathway culminating in RNR-R2 expression and the generation of an active RNR holoenzyme, the key regulator of dNTP levels, leading to an increase in dNTPs. How the virus induces DDR and RNR-R2 upregulation is not completely known. The viral HBx open reading frame (ORF) was believed to trigger this pathway. Unexpectedly, however, we report here that the production of HBx protein is dispensable. We found that a small conserved region of 125 bases within the HBx ORF is sufficient to upregulate RNR-R2 expression in growth-arrested HepG2 cells and primary human hepatocytes. The observed HBV mRNA embedded regulatory element is named ERE. ERE in isolation is sufficient to activate the ATR-Chk1-E2F1-RNR-R2 DDR pathway. These findings demonstrate a non-coding function of HBV transcripts to support its propagation in non-cycling cells.

Viral Decoys: The Only Two Herpesviruses Infecting Invertebrates Evolved Different Transcriptional Strategies to Deflect Post-Transcriptional Editing

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host’s immune response in different virus–host combinations.

Alternative splicing of viral transcripts: the dark side of the HBV

Regulation of alternative splicing is one of the most efficient mechanisms to enlarge the proteomic diversity in eukaryotic organisms. Many viruses hijack the splicing machinery following infection to accomplish their replication cycle. Regarding the HBV, numerous reports have described alternative splicing events of the long viral transcript (pregenomic RNA), which also acts as a template for viral genome replication. Alternative splicing of HBV pregenomic RNAs allows the synthesis of at least 20 spliced variants. In addition, almost all these spliced forms give rise to defective particles, detected in the blood of infected patients. HBV-spliced RNAs have long been unconsidered, probably due to their uneasy detection in comparison to unspliced forms as well as for their dispensable role during viral replication. However, recent data highlighted the relevance of these HBV-spliced variants through (1) the trans-regulation of the alternative splicing of viral transcripts along the course of liver disease; (2) the ability to generate defective particle formation, putative biomarker of the liver disease progression; (3) modulation of viral replication; and (4) their intrinsic propensity to encode for novel viral proteins involved in liver pathogenesis and immune response. Altogether, tricky regulation of HBV alternative splicing may contribute to modulate multiple viral and cellular processes all along the course of HBV-related liver disease.

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Cellular differentiation, which is necessary for HPV life cycle completion disrupts CTCF-dependent chromatin looping of HPV18 episomes inducing enhanced activity of the HPV18 early promoter P105 and increased viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species in these cultures, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells (HPV18-ΔCTCF) identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF of HPV18 promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate importance of CTCF-dependent transcription regulation at multiple stages of the HPV life cycle.

The ENE of polyadenylated nuclear (PAN) RNA is not required for productive lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV)

KSHV is an oncogenic human gammaherpesvirus and the causative agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multi-centric Castleman’s disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode for transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors are expressed that are required for lytic replication. The most abundant viral transcript is the long non-coding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNA have diverse functions, including regulation of gene expression and immune response. PAN possesses two main cis-acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN still remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV BACmid containing a deletion of the 79-nt ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, ORF59, LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlights how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggests that PAN may have other functional domains apart from the ENE. IMPORTANCE KSHV is an oncogenic herpesvirus which establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with lytic replication of the virus. PAN RNA is the most abundant viral transcript during reactivation of KSHV and is required for viral replication. Deletion and knockdown studies of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the cis-elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and in vitro approaches, this is the first study to demonstrate the role of ENE in the context of infection, and that the ENE of PAN is not required for lytic replication of KSHV.

A Tale of Two Transcriptomic Responses in Agricultural Pests via Host Defenses and Viral Replication

Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) is a baculovirus that causes systemic infections in many arthropod pests. The specific molecular processes underlying the biocidal activity of AcMNPV on its insect hosts are largely unknown. We describe the transcriptional responses in two major pests, Spodoptera frugiperda (fall armyworm) and Trichoplusia ni (cabbage looper), to determine the host–pathogen responses during systemic infection, concurrently with the viral response to the host. We assembled species-specific transcriptomes of the hemolymph to identify host transcriptional responses during systemic infection and assessed the viral transcript abundance in infected hemolymph from both species. We found transcriptional suppression of chitin metabolism and tracheal development in infected hosts. Synergistic transcriptional support was observed to suggest suppression of immune responses and induction of oxidative stress indicating disease progression in the host. The entire AcMNPV core genome was expressed in the infected host hemolymph with a proportional high abundance detected for viral transcripts associated with replication, structure, and movement. Interestingly, several of the host genes that were targeted by AcMNPV as revealed by our study are also targets of chemical insecticides currently used commercially to control arthropod pests. Our results reveal an extensive overlap between biological processes represented by transcriptional responses in both hosts, as well as convergence on highly abundant viral genes expressed in the two hosts, providing an overview of the host–pathogen transcriptomic landscape during systemic infection.

Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus

Rotaviruses transcribe eleven distinct types of protein-coding RNAs that must be stoichiometrically co-packaged prior to their replication to make an infectious dsRNA virion. During infection, a fraction of rotavirus transcripts accumulates in the cytoplasmic ribonucleoprotein (RNP) condensates, known as viroplasms. Understanding the mechanisms of viroplasm assembly and RNA enrichment within is crucial to gaining greater insight into their function and stoichiometric assortment of individual transcripts. We analysed the subcellular distribution of individual RV transcripts and viroplasm transcriptome by combining multiplexed DNA-barcoded single-molecule RNA FISH of infected cells. Using DNA-PAINT microscopy, we provide evidence of the early onset of viral transcript oligomerisation that occurs prior to the formation of viroplasms. We demonstrate that viral sequences lacking the untranslated regions (UTRs) fail to undergo enrichment in rotavirus RNP condensates. We show that individual viral transcripts exhibit variable propensities to partition into viroplasms, irrespective of their absolute numbers in cells, suggesting a selective RNA enrichment mechanism distinct from other known cellular RNP granules. We suggest that rotavirus replication factories represent unique RNP condensates enriched in eleven types of cognate transcripts that may facilitate the assembly of a multi-segmented RNA genome.

Bacterial, Archaea, and Viral Transcripts (BAVT) Expression in Gynecological Cancers and Correlation with Regulatory Regions of the Genome

Bacteria, archaea, and viruses are associated with numerous human cancers. To date, microbiome variations in transcription have not been evaluated relative to upper female genital tract cancer risk. Our aim was to assess differences in bacterial, archaea, and viral transcript (BAVT) expression between different gynecological cancers and normal fallopian tubes. In this case-control study we performed RNA sequencing on 12 normal tubes, 112 serous ovarian cancers (HGSC) and 62 endometrioid endometrial cancers (EEC). We used the centrifuge algorithm to classify resultant transcripts into four indexes: bacterial, archaea, viral, and human genomes. We then compared BAVT expression from normal samples, HGSC and EEC. T-test was used for univariate comparisons (correcting for multiple comparison) and lasso for multivariate modelling. For validation we performed DNA sequencing of normal tubes in comparison to HGSC and EEC BAVTs in the TCGA database. Pathway analyses were carried out to evaluate the function of significant BAVTs. Our results show that BAVT expression levels vary between different gynecological cancers. Finally, we mapped some of these BAVTs to the human genome. Numerous map locations were close to regulatory genes and long non-coding RNAs based on the pathway enrichment analysis. BAVTs may affect gynecological cancer risk and may be part of potential targets for cancer therapy.

Profiling the Blood Compartment of Hematopoietic Stem Cell Transplant Patients During Human Cytomegalovirus Reactivation

Human cytomegalovirus (HCMV) is a widespread pathogen establishing a latent infection in its host. HCMV reactivation is a major health burden in immunocompromised individuals, and is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Here we determined HCMV genomic levels using droplet digital PCR in different peripheral blood mononuclear cell (PBMC) populations in HCMV reactivating HSCT patients. This high sensitivity approach revealed that all PBMC populations harbored extremely low levels of viral DNA at the peak of HCMV DNAemia. Transcriptomic analysis of PBMCs from high-DNAemia samples revealed elevated expression of genes typical of HCMV specific T cells, while regulatory T cell enhancers as well as additional genes related to immune response were downregulated. Viral transcript levels in these samples were extremely low, but remarkably, the detected transcripts were mainly immediate early viral genes. Overall, our data indicate that HCMV DNAemia is associated with distinct signatures of immune response in the blood compartment, however it is not necessarily accompanied by substantial infection of PBMCs and the residual infected PBMCs are not productively infected.

Basal Expression of Interferon-Stimulated Genes Drives Population Differences in Monocyte Susceptibility to Influenza Infection

SUMMARYThere is considerable inter-individual immunological and clinical variability upon influenza A virus (IAV) infection in humans; yet, the factors underlying such heterogeneity remain elusive. Here, using an ex vivo cellular model that captures natural human variation in the transcriptional responses of monocytes to IAV, we find significant differences in viral mRNA levels between individuals of African and European ancestry. Using single cell analyses, we show that the overall number of cells that will ultimately become infected, rather than the amount of viral transcript expression per cell, is the main driver of the higher IAV mRNA levels detected in European cells. Finally, we identify 135 genes, including the interferon-stimulated genes IFITM3, MX1, and OAS3, for which basal mRNA expression levels associate with IAV mRNA levels post infection. Our findings reveal that basal differences in activation of IRF/STAT-induced antiviral mechanisms may contribute to individual and population disparities in susceptibility to IAV infection.