Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

Abstract

Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

Single-cell RNA-seq reveals transcriptomic heterogeneity mediated by host–pathogen dynamics in lymphoblastoid cell lines

“…Host genes upregulated within lytic cluster cells (e.g., NFATC1 , MIER2 , SFN , and SGK1 ) represent a limited subset of transcription factors associated with B (and T) lymphocyte activation ( Peng et al, 2001 ; Tsitsikov et al, 2001 ), several of which have been recently identified at various degrees of enrichment within lytic cells ( Frey et al, 2020 ). The presence of NFATC1 is particularly notable considering the recent report of this factor contributing to the spontaneous lytic phenotype of type 2 EBV by upregulating expression of BZLF1 to promote the lytic gene expression cascade ( Romero-Masters et al, 2020 ).…”

“…Host genes upregulated within lytic cluster cells (e.g., NFATC1, MIER2, SFN, SGK1) represent a limited subset of transcription factors associated with B (and T) lymphocyte activation 72,73 , several of which have been recently identified at various degrees of enrichment within lytic cells. 74 The presence of NFATC1 is particularly notable considering the recent report of this factor contributing to the spontaneous lytic phenotype of type 2 EBV by upregulating expression of BZLF1 to promote the lytic gene expression cascade. 75 Although PC loadings reveal substantial upregulation of more than a dozen EBV lytic genes, cells within the lytic clusters curiously lack expression of BZLF1, which plays a role in the latentto-lytic transition.…”

A survey of RNA secondary structural propensity encoded within human herpesvirus genomes: global comparisons and local motifs

“…Here, results can be browsed alongside genomic annotations (which have been loaded from NCBI for each genome) or any JBrowse compatible file ( Buels et al, 2016 ) a user has available. As an example, for EBV-1 we include additional tracks alongside results to help identify regions of interest; the McIntosh lab has generated RNA sequencing data for the EBV-1 genome as it transitions from a latent to a lytically active state ( Frey et al, 2020 ). The sequencing data from this study can now be seen as a coverage track and allows users to quickly assess whether regions highlighted by fall within actively transcribed regions.…”

Peer Review #1 of "A survey of RNA secondary structural propensity encoded within human herpesvirus genomes: global comparisons and local motifs (v0.1)"

“…Here, results can be browsed alongside genomic annotations (which have been loaded from NCBI for each genome) or any JBrowse compatible file (Buels et al 2016) a user has available. As an example, for EBV-1 we include additional tracks alongside ScanFold results to help identify regions of interest; the McIntosh lab has generated RNA sequencing data for the EBV-1 genome as it transitions from a latent to a lytically active state (Frey et al 2020). The sequencing data from this study can now be seen as a coverage tracks and allows users to quickly assess whether regions highlighted by ScanFold fall within actively transcribed regions.…”