Developing a universal strategy for cloning and assembly of the genomes of diverse Epstein – Barr virus strains

Abstract

Epstein-Barr virus (EBV), is an oncogenic gamma-herpesvirus, which is associated with malignant diseases of B cells, T cells, and epithelial cells. EB viruses have large DNA genomes of more than 170 kb that are difficult to clone and manipulate. Here we describe 2 different approaches for cloning whole EBV genomes of diverse strains for reverse genetics studies. The first approach used CRISPR/Cas9-mediated cloning of the entire EBV genome into a bacterial artificial chromosome (BAC) vector using homologous recombination in B cells. This method allowed the cloning of the type 2 EBV strain Jijoye for the first time, but the BAC-clones are unstable. This strategy is being modified by recoding the homology regions to make the clones more stable. The second approach involves transformation-associated recombination (TAR) cloning of EBV fragments and their assembly in yeast, which will allow for mixing and matching DNA regions from different EBV strains for functional studies. This approach is based on TAR cloning of the EBV genome as 10 overlapping fragments, which average 17 kilobases long, using the natural homologous recombination processes of the yeast. Subsequent assembly of all the overlapping fragments is undertaken in yeast or by Gibson assembly to reconstitute the infectious EBV clone. Two fragments from EBV strains B95-8 and AG876 were captured and isolated successfully, but at low efficiency. We are currently improving the TAR cloning efficiency by increasing the size of the capture homology regions to approximately 500 bp coupled with CRISPR/Cas-9-mediated fragmentation of the EBV genome.