Ever since the pioneering work of the Khorana' group that synthesized the 77-bp nucleotide gene encoding a yeast alanine transfer RNA and the 207-bp gene for the tyrosine suppressor tRNA[1, 19] 40 years ago, scientists have had the ability to join DNA sequences and produce combinations that are not present in nature. Recently, chemical synthesis of genes has become routine, and the chemical synthesis of genomes has now become a reality and several viral genomes generated by the chemical synthesis have been reported[4, 20]. Gibson et al. developed a method for assembling the 1.08-mega-base Mycoplasma mycoides genome staring from digitized genome sequence information, and successfully transplanted it into a Mycoplasma capricolum recipient cell to create a modified Mycoplasma mycoides cell controlled by a synthetic genome .
Transfusion Transmitted Virus was first reported as a post-transfusion hepatitis virus of unexplained etiology in Japanese patients in 1997[14, 15] and was recently named as Torque teno virus (TTV) as the first member of the new family Anelloviridae. The virus is a nonenveloped virus containing a single-stranded, circular DNA genome of approximately 3.8 kb [13, 22]. Until now, at least 39 genotypes of TTV have been identified, and they can be classified into five distantly related groups . The TTV genome includes an untranslated region (UTR) of approximately 1.2kb and a coding region of approximately 2.6kb, including two main open reading frames (ORFs) which are sandwiched by the TATA box and polyadenylation signal motifs[13, 14, 16]. Epidemiological studies have shown that TTV is genetically variable and widespread in the general human population. Although replication of TTV indicates that it occurs in the liver , and most researchers have considered that it might have a possible association with non-A-G hepatitis, post-transfusion hepatitis which may induce liver disease [3, 10, 15, 16], to date there is no consistent evidence of a link between TTV infection of humans and specific disease . The virus is extremely common, even in healthy individuals-as much as 100% prevalent in some countries, and in approximately 10% of blood donors in the UK and the US. The exact role of this virus in the pathogenesis of chronic liver diseases remains controversial.
In this study, we obtained the entire 3.8 kb synthetic TTV genome using a purely in vitro process. Through this work, some experience in assembling the genomes containing complex structures from oligonucleotides has been gained, thereby we can reconstruct viral genomes less than 10 kb and accelerate the preparation of live attenuated vaccines. Viruses also represent natural systems which can deliver foreign genes into target cells; therefore, the artificial TTV genome could be developed as a hepatic disease gene therapy vector in the future.
Total Chemical Synthesis, Assembly of Human Torque Teno Virus Genome
- Received Date: 29 January 2011
- Accepted Date: 02 March 2011
Abstract: Torque teno virus (TTV) is a nonenveloped virus containing a single-stranded, circular DNA genome of approximately 3.8kb. We completely synthesized the 3 808 nucleotides of the TTV (SANBAN isolate) genome, which contains a hairpin structure and a GC-rich region. More than 100 overlapping oligonucleotides were chemically synthesized and assembled by polymerase chain assembly reaction (PCA), and the synthesis was completed with splicing by overlap extension (SOEing). This study establishes the methodological basis of the chemical synthesis of a viral genome for use as a live attenuated vaccine or gene therapy vector.