The DNA genomes of herpesviruses range in size from 120 kb to 240 kb and hence, have a large coding capacity in some cases in excess of 100 gene products. For years, genetic manipulation of these genomes was feasible for only a subset of these viruses. Subsequently, many of the herpesvirus genomes were cloned into BAC plasmids, which significantly advanced the technologies of genome engineering in an E.coli host and the successful reconstitution of infectious virus in the appropriate host cell. In this application, we propose a transformational approach, which is to use synthetic biology to build wild-type clones of the Kaposi's sarcoma- associated herpesvirus (KSHV) genome and demonstrate the reconstitution of infectivity of these assembled genomes. The successful outcome of this synthetic genomics approach will significantly advance the ability to clone, assemble and engineer this important virus in a more high-throughput manner.
Specific Aim 1 : Use synthetic genomics methods to clone and assemble KSHV genomes in yeast. In this aim, we will clone the KSHV genomes from two strains, BCBL-1 and JSC-1, using synthetic genomics methods. These genomes will be deconstructed into 11 parts, which can be modified separate of each other and then reassembled using yeast homologous recombination. Our approach will be based on advances made in this field by members of the J. Craig Venter Institute (JCVI) team that created the first synthetic microbe. In a collaborative effort, we have already assembled an infectious clone of herpes simplex virus type-1 (HSV-1) using these methods and are close to completion of an Epstein-Barr virus (EBV) Akata genome. We will use these new and powerful synthetic genomics methods to assemble complete genomes of KSHV in yeast.
Specific Aim 2 : Reconstitute biological activity of the assembled KSHV genomes in mammalian cells. The goal in this aim will be to recover infectious virus after introduction of assembled herpesvirus genomes into mammalian cells. KSHV assembled genomes will be transfected/electroporated endothelial cells (TIME - telomerase-immortalized microvascular endothelial cells) and BJAB cells. Cell lines that harbor the KSHV episome, following drug selection, will be induced for lytic virus production. Biological activity will be measured using latency antigen (LANA) staining to measure establishment of latency as well as spindle cell conversion of endothelial cells. Virus reactivation following lytic activation will be determined using quantitative PCR to measure viral genomes, lytic gene expression as well as TIME GFP titers. Our singular goal is to use the combined and complementary expertise of the JHU and JCVI laboratories to demonstrate we can assemble complete genomes of herpesviruses from the individual parts in an efficient process with high fidelity and stability. If successful, this would provide a new powerful platform to clone and manipulate these viruses to facilitate the study of their biology. This technology will thus, complement and extend the existing BAC methods.
Genetic manipulation of the large herpesvirus DNA genomes has made significant advances using BAC technology. In this application we propose to make a transformational change to this important method by taking advantage of synthetic biology tools. Thus, we will build infectious clones of the wild-type Kaposi's sarcoma-associated herpesvirus genome using synthetic genomics engineering.
