Essential Role of Rta in Lytic DNA Replication of Epstein-Barr Virus

Abstract

f Two transcription factors, ZEBRA and Rta, switch Epstein-Barr virus (EBV) from the latent to the lytic state. While ZEBRA also plays an obligatory role as an activator of replication, it is not known whether Rta is directly required for replication. Rta is dispensable for amplification of an oriLyt-containing plasmid in a transient-replication assay. Here, we assessed the requirement for Rta in activation of viral DNA synthesis from the endogenous viral genome, a function that has not been established. Initially, we searched for a ZEBRA mutant that supports viral replication but not transcription. We found that Z(S186A), a mutant of ZEBRA unable to activate transcription of Rta or viral genes encoding replication proteins, is competent to bind to oriLyt and to function as an origin recognition protein. Ectopic expression of the six components of the EBV lytic replication machinery failed to rescue replication by Z(S186A). However, addition of Rta to Z(S186A) and the mixture of replication factors activated viral replication and late gene expression. Deletion mutagenesis of Rta indicated that the C-terminal 10 amino acids (aa) were essential for the function of Rta in replication. In vivo DNA binding studies revealed that Rta interacted with the enhancer region of oriLyt. In addition, expression of Rta and Z(S186A) together, but not individually, activated synthesis of the BHLF1 transcript, a lytic transcript required for the process of viral DNA replication. Our findings demonstrate that Rta plays an indispensable role in the process of lytic DNA replication. L ytic infection is intrinsic to Epstein-Barr virus (EBV) pathogenesis. Viral particles are synthesized and assembled exclusively during the lytic state. Activation of the lytic cycle gene expression program is the only route for virus propagation. Activation of the lytic program is mediated by two transcription factors, ZEBRA and Rta, encoded by the viral BZLF1 and BRLF1 genes (1-4). Both proteins are required to trigger sequential events that include expression of replication proteins (RPs), viral genome amplification, and synthesis of late structural proteins (1,2,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).A complete linear viral genome contains two copies of the origin of lytic DNA replication (oriLyt), which regulate the process of genome amplification. These oriLyt sequences are ϳ105 kbp apart and are located in the left and right duplicated sequences of the genome (DS L and DS R ) (15). Naturally occurring EBV strains with deletions of one copy of either origin, such as the B95-8 and P3HR1 virus strains, still maintain the capacity to replicate the entire viral genome (16,17). Each origin of replication contains the DNA regulatory elements sufficient to replicate a surrogate oriLyt plasmid in cis (15). Previous studies with oriLyt plasmids characterized three important cis elements present in the DS L origin, also known as BamHI-H oriLyt (18). These cis elements are the upstream and downstream elements, which are essential for genome amplification, and a ...

Identification of Alternative Transcripts Encoding the Essential Murine Gammaherpesvirus Lytic Transactivator RTA

“…We further showed that this organization of gene 50 transcription was conserved in both EBV and KSHV (16). The unexpected ability of the G50DblKo mutant to replicate under some experimental conditions indicated that there must exist alternative mechanisms for driving gene 50 transcription, since RTA is known to be absolutely required for virus replication (this has been shown for EBV, KSHV, and MHV68) (23,(29)(30)(31)(32). To determine if this hypothesis was indeed correct, we performed 5= RACE analysis of RNAs from Vero-Cre, RAW 264.7, and IFN-␣/␤R Ϫ/Ϫ MEFs infected with either wild-type MHV68 or the G50DblKo mutant.…”

Latency of Epstein–Barr virus is disrupted by gain-of-function mutant cellular AP-1 proteins that preferentially bind methylated DNA

“…The AP-1 mutants strongly activate the EBV BRLF1 gene, the product of which, Rta, stimulates transcription of many viral genes and participates in viral DNA replication during the lytic cycle (28,31). A plausible mechanism for the ability of the AP-1 proteins to activate expression of Rta is the acquisition the capacity to bind methylated ZEBRA response elements embedded in the promoter of the BRLF1 gene encoding Rta.…”

Cytomegalovirus UL91 Is Essential for Transcription of Viral True Late (γ2) Genes

“…Nucleocapsid maturation and release is completed over an approximately 24-h time period (17,24). All herpesviruses build a similar DNA replication complex, such that ORF50/RTA plays an analogous organizing role in gammaherpesviruses (25,26). HCMV UL112-113 proteins can replace RTA to activate lytic replication of Kaposi's sarcoma herpesvirus (KSHV, also called HHV-8) (27), suggesting functional conservation between a betaherpesvirus and a gammaherpesvirus.…”