An Epstein-Barr Virus-Encoded Protein Complex Requires an Origin of Lytic Replication In Cis to Mediate Late Gene Transcription

Abstract

Epstein-Barr virus lytic replication is accomplished by an intricate cascade of gene expression that integrates viral DNA replication and structural protein synthesis. Most genes encoding structural proteins exhibit “true” late kinetics–their expression is strictly dependent on lytic DNA replication. Recently, the EBV BcRF1 gene was reported to encode a TATA box binding protein homolog, which preferentially recognizes the TATT sequence found in true late gene promoters. BcRF1 is one of seven EBV genes with homologs found in other β- and γ-, but not in α-herpesviruses. Using EBV BACmids, we systematically disrupted each of these “βγ” genes. We found that six of them, including BcRF1, exhibited an identical phenotype: intact viral DNA replication with loss of late gene expression. The proteins encoded by these six genes have been found by other investigators to form a viral protein complex that is essential for activation of TATT-containing reporters in EBV-negative 293 cells. Unexpectedly, in EBV infected 293 cells, we found that TATT reporter activation was weak and non-specific unless an EBV origin of lytic replication (OriLyt) was present in cis. Using two different replication-defective EBV genomes, we demonstrated that OriLyt-mediated DNA replication is required in cis for TATT reporter activation and for late gene expression from the EBV genome. We further demonstrate by fluorescence in situ hybridization that the late BcLF1 mRNA localizes to EBV DNA replication factories. These findings support a model in which EBV true late genes are only transcribed from newly replicated viral genomes.

A Temporal Proteomic Map of Epstein-Barr Virus Lytic Replication in B Cells

“…The Tp1 cluster contains multiple members of the EBV viral preinitiation complex, which activates expression of most EBV late genes from newly replicated viral DNA (Aubry et al., 2014, Djavadian et al., 2016). The Tp2 cluster included proteins important in inhibition of host innate immunity, and multiple tegument, capsid, and envelope proteins were present in the later-expressed Tp3 class.…”

The interaction between ORF18 and ORF30 is required for late gene expression in Kaposi’s sarcoma-associated herpesvirus

“…The fact that the late gene expression defect of the ORF18 E36A_L37A mutant is exacerbated in the context of the virus, compared to in the plasmid promoter activation assay, likely reflects the fact that the plasmid assay measures basal promoter activation but misses other regulatory components of this cascade. For example, the origin of lytic replication is required in cis for late gene expression in related gammaherpesviruses (3, 28) and the reporter assay does not capture the important contribution of viral DNA replication towards late gene expression. This may explain why some mutants that are defective for ORF30 binding (e.g.…”

The Epstein-Barr Virus Immunoevasins BCRF1 and BPLF1 Are Expressed by a Mechanism Independent of the Canonical Late Pre-initiation Complex

“…These proteins are: BcRF1, BDLF3.5, BDLF4, BFRF2, and BVLF1. Knockout of BcRF1, BDLF4, BVLF1, BDLF3.5 and BFRF2 disrupts expression of late genes [11–14]. However, the exact function of several of these late gene regulators is still unknown.…”

“…HCMV UL79, the ortholog of EBV BVLF1, functions as an elongation factor of RNAPII during the late phase of viral gene expression [20]. Sub-nuclear localization studies revealed that late gene regulators assemble in replication compartments and that true late genes are transcribed from newly replicated viral DNA [14, 21, 22]. Formation of replication compartments might increase the local concentration of these late gene regulators and promote their interaction with replication proteins to stimulate synthesis of late transcripts.…”