Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

Abstract

The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation. Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that infects epithelial and lymphoid cells and is associated with several human malignancies (for a review, see reference 59).EBV undergoes lytic replication in epithelial cells during primary infection and during reactivation from latent infection in B lymphocytes. The switch from latency to lytic replication is regulated by two EBV transcriptional activators: Z (also known as BZLF1, Zta, or ZEBRA) and R (also known as BRLF1 or Rta) (for a review, see reference 68). SM (also known as Mta, EB2, or BMLF1) is an RNA-binding EBV protein that is expressed following Z and R and enhances EBV gene expression by multiple posttranscriptional mechanisms (4,11,28,29,35,61,65). SM is essential for the lytic phase of EBV replication and virion production (21). We previously used an epithelial cell line carrying an EBV recombinant deleted for SM (293 BMLF1-KO) to compare gene expression in the presence and absence of SM (25). When cells carrying recombinant SM-deleted EBV were transfected with the EBV transactivator BZLF1 gene, approximately 50% of EBV genes were expressed poorly but were rescued by cotransfection with SM, resulting in EBV DNA replication and virion production (25). The block in EBV DNA replication is at least partly due to inefficient expression of EBV DNA polymerase and primase in the absence of the SM protein. Transfection of EBV DNA polymerase and primase genes with BZLF1 allows EBV DNA replication but does not rescue virion production, indicating that additional essential EBV mRNAs directly require SM for efficient expression (25).When EBV lytic replication was rescued by SM transfection, expression of most EBV genes either was upregulated or was not significantly changed, but BHRF1 mRN...

General and Target-Specific RNA Binding Properties of Epstein-Barr Virus SM Posttranscriptional Regulatory Protein

“…The RNA in those studies was detected by a sensitive RNase protection assay, and it was unclear whether IP of SM from EBV-infected cells would yield associated RNA in amounts adequate to be analyzed by qRT-PCR for all EBV transcripts. We therefore utilized a cell line derived from the Burkitt lymphoma cell line, P3HR1, that was engineered to permit efficient, high-level lytic EBV replication and gene expression (45). The P3HR1-ZHT cell line contains a stably transfected chimeric gene consisting of the Z immediate-early transactivator fused to a 4-HT-specific hormone binding domain.…”

“…P3HR1 is a Burkitt lymphoma cell line infected with EBV type 2 (31), and B95-8 is a marmoset B-cell line transformed by EBV type 1 (26). Both cell lines were transfected and selected to stably express a fusion protein containing the BZLF1 transactivator of early lytic cycle replication fused to the hormone domain of the estrogen receptor, which, in the presence of 4-hydroxytamoxifen (4-HT), allows the functional release of BZLF1, inducing lytic EBV replication (19,45). The P3HR1-ZHT and B958-ZHT cell lines were propagated in RPMI supplemented with 10% fetal bovine serum (HyCLone), 0.8 mg/ml G418 (AG Scientific), and GlutaMax (Invitrogen).…”

Spironolactone blocks Epstein–Barr virus production by inhibiting EBV SM protein function

“…HEK293 cells stably carrying an ORF59-GFP fusion gene that also inducibly expresses SM-dependent GFP were maintained as previously described (8). P3HR1-ZHT is an EBV-positive lymphoma cell line that contains the EBV BZLF1 gene fused to the hormone-binding domain of the 4-hydroxytamoxifen (4HT) receptor that allows robust induction of EBV replication with 4HT treatment (30). The gastric carcinoma cell line AGS was infected with GFP-expressing EBV Akata BX1 (31).…”

Epstein-Barr Virus SM Protein Utilizes Cellular Splicing Factor SRp20 To Mediate Alternative Splicing

“…We and others have previously shown that SM plays multiple roles in posttranscriptional processing of EBV mRNA and cellular mRNA (2,9,17,18,23,31,35,39,40). Although the target sequences for SM binding have not been biochemically defined, abundant evidence indicates that SM contacts RNA directly.…”

“…SM has multiple functions in enhancing EBV gene expression posttranscriptionally, binds target gene mRNA, enhances nuclear mRNA export and stability, and modulates cellular and EBV RNA splicing (2,9,17,18,23,31,35,39,40). SM is essential for EBV replication and EBV recombinants with insertional deletion of the SM gene are defective for virus production (12).…”