Accurate quantification of overlapping herpesvirus transcripts from RNA-seq data

Herpesviruses employ extensive bidirectional transcription of overlapping genes to overcome length constraints on their gene product repertoire. As a consequence, many lytic transcripts cannot be measured individually by RT-qPCR or conventional RNA-seq analysis. Bruce et al. (Pathogens 2017, 6, 11; doi:10.3390/pathogens6010011) proposed an approximation method using Unique CoDing Sequences (UCDS) to estimate lytic gene abundance from KSHV RNA-seq data. Although UCDS has been widely employed, its accuracy, to our knowledge, has never been rigorously validated for any herpesvirus. In this study, we use CAGE-seq as a gold-standard to determine the accuracy of UCDS for estimating EBV lytic gene expression levels from RNA-seq data. We also introduce the Unique TranScript (UTS) method that, like UCDS, estimates transcript abundance from changes in mean RNA-seq read-depth. UTS is distinguished by its use of empirically determined 5’ and 3’ transcript ends, rather than coding sequence annotations. Compared to conventional read assignment, both UCDS and UTS improved quantitation accuracy of overlapping genes, with UTS giving the most accurate results. The UTS method discards fewer reads and may be advantageous for experiments with less sequencing depth. UTS is compatible with any aligner and, unlike isoform-aware alignment methods, can be implemented on a laptop computer. Our findings demonstrate that accuracy achieved by complex and expensive techniques such as CAGE-seq can be approximated using conventional short-read RNA-seq data when read assignment methods address transcript overlap. Although our study focuses on EBV transcription, the UTS method should be applicable across all herpesviruses and other genomes with extensively overlapping transcriptomes. IMPORTANCE Many viruses employ extensively overlapping transcript structures. This complexity makes it difficult to quantify gene expression using conventional methods including RNA-seq. Although high-throughput techniques that overcome these limitations exist, they are complex, expensive, and scarce in herpesvirus literature relative to short-read RNA-seq. Here, using Epstein-Barr virus (EBV) as a model, we demonstrate that conventional RNA-seq analysis methods fail to accurately quantify abundance of many overlapping transcripts. We further show that the previously described Unique CoDing Sequence (UCDS) and our Unique TranScript (UTS) methods greatly improve the accuracy of EBV lytic gene measurements obtained from RNA-seq data. The UTS method has the advantages of discarding fewer reads and being implementable on a laptop computer. Although this study focuses on EBV, the UCDS and UTS methods should be applicable across herpesviruses and for other viruses that make extensive use of overlapping transcription.

Arsenicals, the Integrated Stress Response, and Epstein–Barr Virus Lytic Gene Expression

Following our observation that clofoctol led to Epstein–Barr virus (EBV) lytic gene expression upon activation of the integrated stress response (ISR), we decided to investigate the impact of As2O3 on viral lytic gene expression. As2O3 has also been reported to activate the ISR pathway by its activation of the heme-regulated inhibitor (HRI). Our investigations show that As2O3 treatment leads to eIF2α phosphorylation, upregulation of ATF4 and TRB3 expression, and an increase of EBV Zta gene expression in lymphoid tumor cell lines as well as in naturally infected epithelial cancer cell lines. However, late lytic gene expression and virion production were blocked after arsenic treatment. In comparison, a small molecule HRI activator also led to increased Zta expression but did not block late lytic gene expression, suggesting that As2O3 effects on EBV gene expression are also mediated through other pathways.

The Role of Lytic Infection for Lymphomagenesis of Human γ-Herpesviruses

Epstein Barr virus (EBV) and Kaposi sarcoma associated herpesvirus (KSHV) are two oncogenic human γ-herpesviruses that are each associated with 1-2% of human tumors. They encode bona fide oncogenes that they express during latent infection to amplify their host cells and themselves within these. In contrast, lytic virus particle producing infection has been considered to destroy host cells and might be even induced to therapeutically eliminate EBV and KSHV associated tumors. However, it has become apparent in recent years that early lytic replication supports tumorigenesis by these two human oncogenic viruses. This review will discuss the evidence for this paradigm change and how lytic gene products might condition the microenvironment to facilitate EBV and KSHV associated tumorigenesis.

KDM2B Overexpression Facilitates Lytic De Novo KSHV Infection by Inducing AP-1 Activity Through Interaction with the SCF E3 Ubiquitin Ligase Complex

It is still largely unknown what host factors are involved in controlling the expression of the lytic viral gene RTA during primary infection, which determines if Kaposi’s sarcoma-associated herpesvirus (KSHV) establishes latent or lytic infection. We have recently identified the histone demethylase KDM2B as a repressor of RTA expression during both de novo KSHV infection and latency based on an epigenetic factor siRNA screen. Here, we report that surprisingly, KDM2B overexpression can promote lytic de novo infection by using a mechanism that differs from what is needed for its repressor function. Our study revealed that while the DNA-binding and demethylase activities of KDM2B linked to its transcription repressive function are dispensable, its C-terminal F-box and LRR domains are required for the lytic infection-inducing function of KDM2B. We found that overexpressed KDM2B increases the half-life of the AP-1 subunit c-Jun protein and induces the AP-1 signaling pathway. This effect is dependent upon the binding of KDM2B to the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex via its F-box domain. Importantly, the inhibition of AP-1 reduces KDM2B-mediated lytic de novo KSHV infection. Overall, our findings indicate that KDM2B may induce the degradation of some host factors by using the SCF complex resulting in the enrichment of c-Jun. This leads to increased AP-1 transcriptional activity, which facilitates lytic gene expression following de novo infection interfering with the establishment of viral latency. Significance The expression of epigenetic factors is often dysregulated in cancers or upon specific stress signals, which often results in a display of non-canonical functions of the epigenetic factors that are independent from their chromatin-modifying roles. We have previously demonstrated that KDM2B normally inhibits KSHV lytic cycle using its histone demethylase activity. Surprisingly, we found that KDM2B overexpression can promote lytic de novo infection, which does not require its histone demethylase or DNA-binding functions. Instead, KDM2B uses the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex to induce AP-1 transcriptional activity, which promotes lytic gene expression. This is the first report that demonstrates a functional link between SFCKDM2B and AP-1 in the regulation of KSHV lytic cycle.

Direct Evidence of Abortive Lytic Infection-Mediated Establishment of Epstein-Barr Virus Latency During B-Cell Infection

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and antiviral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, we infected EBV-negative Akata cells with EBV-EGFP and performed transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprogramming of cell growth by EBV, and a burst of viral lytic gene expression in the early phase of infection. Experimental and mathematical investigations demonstrate that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Fate mapping using recombinant EBV provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency. Furthermore, we find that the BZLF1 protein, which is a key regulator of reactivation, was dispensable for abortive lytic infection in the pre-latent phase, suggesting the divergent regulation of viral gene expressions from a productive lytic infection.

Epstein–Barr Virus: How Its Lytic Phase Contributes to Oncogenesis

Epstein–Barr Virus (EBV) contributes to the development of lymphoid and epithelial malignancies. While EBV’s latent phase is more commonly associated with EBV-associated malignancies, there is increasing evidence that EBV’s lytic phase plays a role in EBV-mediated oncogenesis. The lytic phase contributes to oncogenesis primarily in two ways: (1) the production of infectious particles to infect more cells, and (2) the regulation of cellular oncogenic pathways, both cell autonomously and non-cell autonomously. The production of infectious particles requires the completion of the lytic phase. However, the regulation of cellular oncogenic pathways can be mediated by an incomplete (abortive) lytic phase, in which early lytic gene products contribute substantially, whereas late lytic products are largely dispensable. In this review, we discuss the evidence of EBV’s lytic phase contributing to oncogenesis and the role it plays in tumor formation and progression, as well as summarize known mechanisms by which EBV lytic products regulate oncogenic pathways. Understanding the contribution of EBV’s lytic phase to oncogenesis will help design ways to target it to treat EBV-associated malignancies.

The KSHV ORF20 Protein Interacts With The Viral Processivity Factor ORF59 And Promote Viral Reactivation

Upon KSHV lytic reactivation, rapid and widespread amplification of viral DNA (vDNA) triggers significant nuclear reorganization. As part of this striking shift in nuclear architecture, viral replication compartments are formed as sites of lytic vDNA production along with remarkable spatial remodeling and relocalization of cellular and viral proteins. These viral replication compartments house several lytic gene products that coordinate viral gene expression, vDNA replication, and nucleocapsid assembly. The viral proteins and mechanisms that regulate this overhaul of the nuclear landscape during KSHV replication remain largely unknown. KSHV’s ORF20 is a widely conserved lytic gene among all herpesviruses suggesting it may have a fundamental contribution to the progression of herpesviral infection. Here, we utilized a promiscuous biotin ligase proximity labeling method to identify the proximal interactome of ORF20, which includes several replication-associated viral proteins, one of which is ORF59, the KSHV DNA processivity factor. Using co-immunoprecipitation and immunofluorescence assays, we confirmed the interaction between ORF20 and ORF59 and tracked the localization of both proteins to KSHV replication compartments. To further characterize the function of ORF20, we generated an ORF20-deficient KSHV and compared its replicative fitness relative to wild type virus. Virion production was significantly diminished in the ORF20-deficient virus as observed by supernatant transfer assays. Additionally, we tied this defect in viable virion formation to a reduction in viral late gene expression. Lastly, we observed an overall reduction in vDNA replication in the ORF20-deficient virus implying a key role for ORF20 in the regulation of lytic replication. Taken together, these results capture the essential role of KSHV ORF20 in progressing viral lytic infection by regulating vDNA replication alongside other crucial lytic proteins within KSHV replication compartments.

Reactivation of Herpes Simplex Virus (HSV) from Latency in Response to Neuronal Hyperexcitability

Herpes Simplex Virus (HSV) establishes a latent infection in neurons, in which viral transcription is restricted and viral promoters are associated with heterochromatin. In response to certain stimuli, the virus reactivates to permit transmission. The exact physiological triggers of reactivation, the cell signaling pathways involved, and how signals act on heterochromatin-associated lytic promoters, are not understood. Previously, we identified a role for a neural stress pathway involving DLK and JNK activity in HSV reactivation, triggered by nerve growth factor (NGF) deprivation. Reactivation was associated with a JNK-dependent histone phospho/methyl switch on lytic gene promoters. Because the same histone phospho/methyl switch occurs in cortical neurons following hyperexcitability (triggered by forskolin), we examined whether HSV reactivation was linked to hyperexcitability, and the contribution of JNK activity and histone phosphorylation. Using our primary neuronal model of HSV reactivation, we found that forskolin triggered DLK/JNK-dependent reactivation via a pathway that was distinct from NGF deprivation. The initial burst of HSV lytic gene expression in response to forskolin occurred independently of histone demethylase activity, and was accompanied by a histone phospho/methyl switch. To determine whether forskolin-mediated reactivation was linked to neuronal activity, we investigated the contribution of ion channel activity. Inhibition of voltage-gated potassium and sodium channels, or hyperpolarization-activated cyclic nucleotide-gated channels, prevented forskolin-mediated reactivation. In addition, hyperexcitability, resulting from the removal of a tetrodotoxin block, triggered HSV reactivation in a DLK/JNK-dependent manner. We next investigated whether physiological triggers induce HSV reactivation via hyperexcitability. IL-1 induced DNA damage associated with hyperexcitability in adult neurons. IL-1 also triggered DLK/JNK-dependent HSV reactivation, which was dependent on ion channel activity. Therefore, these data indicate that neuronal hyperexcitability in response to physiological stimuli, such as inflammation, trigger HSV reactivation, and mark out the activation of DLK/JNK and a histone phospho/methyl switch as key events in the hyperexcitability-mediated reactivation.

Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and anti-viral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, EBV-negative Akata cells were infected with EBV-EGFP and observed by transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprograming of cell growth by EBV. Moreover, a burst of viral lytic gene expression was detected in the early phase of infection. Experimental and mathematical investigations demonstrated that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Finally, we conducted fate mapping using recombinant EBV, enabling the noninvasive, continuous observation of infected cells during EBV infection. Our tracking analysis provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency.Author summaryViral infection is a complex process that activates both virus-triggered and host anti-viral responses. This process has classically been studied by snapshot analysis such as microarray and RNA-seq at discrete time points as population averages. Snapshot data lead to invaluable findings in host-pathogen interactions. However, these “snapshot” omics, even from a single cell, lack temporal resolution. Because the behavior of infected cells is highly dynamic and heterogenous, continuous analysis is required for deciphering the fate of infected cells during viral infection. Here, we exploited fate mapping techniques with recombinant Epstein-Barr virus (EBV) to track the infected cells and recorded a log of lytic gene expression during EBV infection. Our continuous observation of infected cells revealed that EBV established latency in B-cells via an abortive lytic infection in the pre-latent phase.