Synthetic herpesvirus genomes with an expanded genetic code

Synthetic biologists re-design biological systems to accelerate fundamental and applied research. Building upon a foundation in herpesvirus research in my laboratory and leadership of the Dalhousie iGEM synthetic biology team, we will use synthetic biology techniques to create a new platform for mutagenesis of human herpesvirus-8 (HHV8). Current approaches for HHV8 genome mutagenesis are inefficient, and complicated by compact viral genome architecture, with many genes closely-associated or overlapping, and transcribed from both DNA strands. In collaboration with the J. Craig Venter Institute and Johns Hopkins University we are assembling a new synthetic HHV8 genome by capitalizing on efficient homologous recombination in yeast. This approach will enable efficient, simultaneous re-coding of herpesvirus genomes at multiple loci, as well as substitution of large segments of the genome with synthetic DNA. This new platform for assembling and mutating virus genomes in yeast will accelerate mapping of essential loci and elucidation of gene function. Only a handful of HHV8 proteins have been thoroughly characterized in the context of viral infection. To overcome this, we will expand the HHV8 genetic code to enable efficient site-specific incorporation of non-canonical amino acids (ncAAs) with useful properties into HHV8 proteins. Our first objective will be to re-code the HHV8 genome in yeast, substituting all 29 mapped amber (TAG) stop codons with alternative TAA or TGA stop codons. This will liberate TAG codons for incorporation of ncAAs into viral proteins. We will confirm that HHV8 viruses bearing this re-coded genome display normal gene expression and replication kinetics. Using this revised genome template, we will create a collection of genomes with TAG codons inserted directly downstream of each annotated translation start site. Thus, in normal cells lacking ncAAs, each recombinant virus will essentially be a single-gene knockout; addition of a cognate ncAA matched to the orthogonal aminoacyl-tRNA synthetase/tRNA pair will allow ncAA incorporation into the targeted protein. This collection will allow us to map essential loci and create new ncAA-containing protein products of these loci that will aid structural and functional protein characterization. Specifically, this system will allow us to incorporate ncAAs with intrinsic fluorescence that will allow us to monitor subcellular localization of viral proteins and larger structures like capsids, facilitating studies of viral assembly and egress. Using the principles of synthetic biology, our NSERC research program will create previously unavailable resources to be widely shared for viral genome manipulation and investigation of viral protein function.