EXPRESSION AND REGULATION OF EBV DNA POLYMERASE

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The Epstein-Barr Virus DNA polymerase gene and its regulation are pivotal not only for replication of the virus, but also in the reactivation cascade from the latent infection state. We are the principal laboratory studying both the gene itself and regulation of its expression. Long-term objectives are to work out the genetics of EBV pol function and drug resistance and to study how expression of the EBV pol in its suppressed and activated states contributes to latency and viral reactivation. In work done in the past 2 years we are the first to show 1) that the EBV BALF 5 ORF encodes active core enzyme, shown both in vitro and in E. coli expression systems. 2) We have mapped the mRNA start-site for the gene by four different methods, and we have identified the EBV pol promoter showing that it is TATA-less and yet not constitutively active. 3) We have also demonstrated transactivation of the promoter by EBV immediate-early genes that act synergistically. 4) Finally and most importantly, in mapping the 3'end of the pol mRNA, we have shown that the pol gene lacks a canonical polyadenylation signal. In its place there appears to be a quite novel mechanism with polyadenylation of the pol mRNA accomplished through a cryptic signal that appears to be recognized by a virally encoded or induced replicative function. The proposed work is organized into three parts and builds on each of these areas, focusing in part one on mapping of catalytic domains of the enzyme and mapping of drug-resistance loci as well as study of a long-suspected EBV pol cofactor, the BMRF-1 product, which is thought to be a processivity factor. The second part dissects the EBV pol promoter in order to define cis-acting regulatory elements and the transactivators needed to activate this ordinarily silent gene. The third part is directed at defining the structure and sequence of the new polyadenylation signal contained in the EBV pol mRNA 3'UTR and in defining the exact sequence needed for this new function. Coupled to this work are experiments to show whether an EBV early trans-acting protein, BMLF-1, which we have already shown acts post-transcriptionally perhaps by stabilizing mRNA, targets the pol message, specifically the sequence in the 3'UTR. This last part of the work is designed to define both a hitherto unrecognized mode of polyadenylation of viral messages as well as a virally encoded gene product which may act as a regulatory or processing protein that mediates polyadenylation.