Genetic Biases in Viral Genomes and the Development of Synthetic Attenuated Virus Engineering (SAVE)

The basic mechanism of mRNA translation is preserved throughout the living world, from the simplest virus through the most complex organism. Viruses, just like human cells, need to produce mRNA molecules to convert their genetic information into protein. Invariably, viruses need to divert the host's cellular machinery for the translation of their proteins, as they themselves cannot execute this function. The degeneracy in the genetic code (several synonymous codons specify the same amino acid) gives an organism the flexibility to encode a given protein sequence within its genome in an unimaginably large number of ways. The HIV Pol protein, for instance, could be encoded by a staggering 10^524 different mRNA sequences, all of them specifying the same protein sequence (for comparison, the estimated number of atoms in the Universe is 10^80). This raises the question to what extent the natural encoding of a protein is optimal or special. The cell's preference for one codon over another to specify the same amino acid is termed "codon bias". It is thought that codon bias correlates with the abundance of the cognate tRNAs in the cell. Consequently, rare codons are associated with suboptimal translation of an mRNA. In addition, the frequencies with which two synonymous codons occur next to one another in an mRNA can differ greatly from what would be statistically expected based on the frequencies of the two codons that make up the codon pair - a phenomenon called the "codon pair bias". Certain codon-pair combinations are statistically greatly underrepresented while others are greatly overrepresented. The biological significance of codon pair bias has been largely unknown and underappreciated. We have recently shown in the poliovirus system that it is possible to exploit the codon pair bias phenomenon for the synthesis of novel live attenuated forms of viruses (Coleman, et al., 2008). By large-scale computer-aided redesign of the viral genome we engineered hundreds of silent mutations into poliovirus. These mutations were targeted to introduce a maximum number of unfavorable synonymous codon-pairs, without changing codon bias or protein sequence. By forcing a virus to "make do" with one of these negatively biased ("de-humanized") synthetic genomes we showed that viral protein translation is greatly reduced. Thus, codon pair-deoptimized viruses cannot reproduce their genetic information as quickly as their wild type cousins, which puts them at a decisive disadvantage against the host's innate and immune defenses. One of the major benefits of the whole-genome deoptimization strategy is that the resulting attenuated (att) viruses are phenotypically and genotypically extremely stable. The att phenotype in "de-humanized" viruses is dependent on many hundreds, even thousands, of silent mutations (each by themselves virtually inconsequential) - resulting in a "death by a thousand cuts". Therefore, the fitness gain from reverting individual mutations appears to be too small to drive active genetic selection, and thus, reversion apparently does not occur (Coleman et al., 2008). We termed this process of perturbing intrinsic viral genome biases by synthetic genome re-design SAVE for Synthetic Attenuated Virus Engineering.

Coleman, et al. 2008

Using the SAVE method of "de-humanizing" viral genomes we profit from these naturally evolved genomic biases and turn them "upside down and inside out", undoing eons of viral evolution, to the detriment of the virus. Since it is evident that virtually all viruses have actively selected against the occurrence of unfavorable codons, codon-pairs, as well as CpG dinucleotides, the whole genome recoding approach is expected to have a profound effects in many virus systems.

In considering these phenomena of genomic biases we suspect that recoding the viral genome according to the SAVE algorithm will perform double duty, 1) by interfering with viral protein translation and, by extension, with the whole life cycle of the virus, and 2) by increasing the viral genome's features of "foreignness" thus increasing its innate "footprint". The resulting viruses are attenuated because they are easier to contain by the host, and they may stimulate better innate and adaptive immunity.

Combining elements of bioinformatics with synthetic biology and "wet lab" virology we are now extending our technology to other virus systems, such as Influenza and HIV, in order to develop novel live attenuated vaccine candidates.