Abstract
Epstein-Barr virus (EBV) lytic replication proceeds through an ordered cascade of gene expression that integrates lytic DNA amplification and late gene transcription. We and others previously demonstrated that 6 EBV proteins that have orthologs in β- and γ-, but not in α-herpesviruses, mediate late gene transcription in a lytic DNA replication-dependent manner. We proposed a model in which the βγ gene-encoded viral pre-initiation complex (vPIC) mediates transcription from newly replicated viral DNA. While this model explains the dependence of late gene transcription on lytic DNA replication, it does not account for this dependence in α-herpesviruses nor for recent reports that some EBV late genes are transcribed independently of vPIC. To rigorously define which transcription start sites (TSS) are dependent on viral lytic DNA replication or the βγ complex, we performed Cap Analysis of Gene Expression (CAGE)-seq on cells infected with wildtype EBV or EBV mutants defective for DNA replication, βγ function, or lacking an origin of lytic replication (OriLyt). This approach identified 16 true-late, 32 early, and 16 TSS that are active at low levels early and are further upregulated in a DNA replication-dependent manner (leaky late). Almost all late gene transcription is vPIC-dependent, with BCRF1 (vIL10), BDLF2, and BDLF3 transcripts being notable exceptions. We present evidence that leaky late transcription is not due to a distinct mechanism, but results from superimposition of the early and late transcription mechanisms at the same promoter. Our results represent the most comprehensive characterization of EBV lytic gene expression kinetics reported to date and suggest that most, but not all EBV late genes are vPIC-dependent.
Highlights
Epstein-Barr virus (EBV) is a γ-herpesvirus that infects more than 95% of the human adult population
We demonstrated that viral pre-initiation complex (vPIC) can only mediate late gene expression when the EBV origin of replication (OriLyt) is present in cis and proposed a model in which EBV vPIC mediates late gene expression by recruiting RNA polymerase II to use newly replicated viral DNA as the template for transcription [30]
We present a comprehensive analysis of the kinetics of EBV lytic gene transcription based on dependence on lytic DNA replication and expression of BDLF4 (a βγ gene encoding an essential component of the viral late gene pre-initiation complex [30,31])
Summary
Epstein-Barr virus (EBV) is a γ-herpesvirus that infects more than 95% of the human adult population. A key advance in our understanding of late gene transcription was the discovery that β- and γ-herpesviruses transcribe their late genes by a mechanism fundamentally different than the α-herpesviruses This discovery began with the finding that several genes with orthologs found only in β- and γ-herpesviruses (βγ genes) were essential for late gene expression, but dispensable for DNA replication [20,21,22,23,24,25,26,27]. One of these βγ genes was shown to encode a viral TATA binding protein (vTBP) [28,29]. We demonstrated that vPIC can only mediate late gene expression when the EBV origin of replication (OriLyt) is present in cis and proposed a model in which EBV vPIC mediates late gene expression by recruiting RNA polymerase II to use newly replicated viral DNA as the template for transcription [30]
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