Abstract
Codon pair bias deoptimization (CPBD) has been successfully used to attenuate several RNA viruses. CPBD involves recoding a viral protein-coding sequence to maximize the number of codon pairs that are statistically underrepresented in the host, which presumably slows protein translation and, hence, causes virus attenuation. However, since recoding preserves the amino acid composition and codon bias, attenuated and parental viruses are antigenically identical. To determine if Marek's disease virus (MDV), a highly oncogenic herpesvirus of the chicken with a large double-stranded DNA genome, can be attenuated by CPBD of its major oncogene meq, we recoded the gene to minimize (meq-D), maximize (meq-O), or preserve (meq-R) the level of codon pairs that are overrepresented in the chicken protein-coding sequences. Unexpectedly, mutants carrying recoded genes produced comparable or increased levels of Meq in the context of viral infection in cultured cells. In addition, parental virus and mutant viruses carrying recoded meq genes replicated with comparable kinetics in vitro and in vivo, and were equally virulent in susceptible chickens. In summary, CPBD of meq failed to produce any quantifiable attenuation of MDV and confirms differences in the complexity of applying CPBD to large DNA viruses versus RNA viruses.
Highlights
Codon pair bias deoptimization (CPBD) is a highly efficient method for virus attenuation
The exact mechanism behind CPBD-mediated virus attenuation is currently explained by two competing, yet poorly understood and unproven, theories: (i) CPBD increases the number of naturally underrepresented codon pair combinations, which results in inefficient translation and reduced protein production [3,4,5]. (ii) CPBD inadvertently increases the number of CpG and TpA dinucleotides in the sequence enabling the recoded viruses to be recognized and cleared by a yet to be identified innate immune mechanism [6, 7]
Our goal was to study the effect of CPBD of meq, one of the non-essential genes of Marek’s disease virus (MDV), on disease progression and pathogenesis. meq is the major oncogene of MDV, an important immunogen as well as one of the few genes that are consistently expressed during lytic replication, latency and transformation [25, 26]
Summary
Codon pair bias deoptimization (CPBD) is a highly efficient method for virus attenuation. CPBD-based attenuation involves reshuffling of available codons in a viral gene to minimize the number of codon pairs that are overrepresented in the protein-coding sequences of the host. The codon pair bias (CPB) of the recoded gene is altered, while the amino acid sequence remains identical to that of the parental protein [3]. Designing CPBD-based vaccines is a matter of minutes and synthetic production a matter of weeks, making CPBD one of the most rapid methods available for virus attenuation. The exact mechanism behind CPBD-mediated virus attenuation is currently explained by two competing, yet poorly understood and unproven, theories: (i) CPBD increases the number of naturally underrepresented codon pair combinations, which results in inefficient translation and reduced protein production [3,4,5]. CPBD-based vaccine candidates for influenza A virus have shown up to 100000-fold attenuation compared to the virulent parental virus and were highly protective in mice and ferrets [14,15,16]
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