Capsid Assembly and Dna Encapsidation by Epstein-Barr Virus(2/3)

Maxi-EBV is a laboratory-constructed Epstein-Barr virus (EBV) strain that contains an inserted F replicon. The insertion does not influence the latent or lytic functions of the virus, so the strain can be maintained and genetically manipulated in E. coli and then analyzed in human cells. In this work, maxi-EBV is mutagenized using a bacterial transposon, EZ::TN＜KAN-2＞, and a PCR-targeting method that involves ？HHf recombinase. Accordingly, a mutant library of 251 mutants with mutations that span the entire EBV genome, including all the annotated EBV genes and the intergenic regions, is established. Studying these mutants revealed that a mutation in BBLF1, BBRF2, BcLF1, BcRF1, BFLF2, BMRF1, BVRF1 or BZLF1 results in strains that cannot generate viral particles after lytic induction, indicating the importance of these genes in the lytic development of EBV. Meanwhile, microarray and immunoblot analyses revealed different roles of Rta and Zta in EBV lytic development. Zta is crucial to the lytic development of EBV because without Zta, almost no lytic genes are expressed in the lytic stage. Rta, however, participates in a different role in lytic development. In the absence of Rta, the productive expression of EBV is inefficient and not sustained. This work also finds that the expression of Rta and Zta depend on each other. Finally, expression of gp350/220 depends not only Rta but also on Zta. This work demonstrates the usefulness of a comprehensive mutant library in genetic analyses of EBV.

Project ID:PG9503-2357
External Project ID:NHRI-EX95-9417BI