Publication Database

Codon pair bias deoptimization of a major oncogene as an attenuation strategy for Marek’s disease herpesvirus (2018)

Art

Hochschulschrift

Autor

Khedkar, Pratik Hemant (WE 5)

Quelle

Berlin, 2018 — 104 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/22970

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Degeneracy of the genetic code enables the encoding of most amino acids by more than one codon. Consequently, there is a vast number of ways in which a protein can be encoded. For example, a peptide with 300 amino acid residues can be encoded in 10^151 different ways. However, in actual coding sequences, the usage of synonymous codons is biased, that is some codons occur more often than their synonymous counterparts, a phenomenon called codon bias. Similarly, but independently of codon bias, occurrence of codon pair combinations in open reading frames (ORFs) is also biased. That is, certain codon pairs appear in coding sequences more often than it would be expected based on the overall frequencies of their constituent codons. This phenomenon, dubbed codon pair bias (CPB), has been found in all the species studied so far and has a greater impact on translational efficiency than codon bias. Virus attenuation by CPB deoptimization (CPBD) is achieved by reshuffling the existing synonymous codons in an ORF without changing the amino acid composition of the encoded protein. The reshuffling thus retains codon bias while simultaneously changing the CPB of the mutated genes. The mechanism behind CPBD mediated attenuation of viruses is currently explained by two competing, however poorly understood, theories: (i) CPBD increases the number of naturally underrepresented codon pair combinations, which results in inefficient translation leading to reduced protein production, and consequently virus attenuation. (ii) CPBD inadvertently increases the number of CpG and TpA dinucleotides in the sequence enabling the recoded viruses to be recognized by a yet to be identified innate immune mechanism. Despite the unclear underlying mechanism, CPBD based attenuation strategy has been successfully employed to attenuate several unrelated RNA viruses, e.g. poliovirus, influenza A virus, dengue virus, etc. The resultant attenuated virus is not only antigenically identical to the parental pathogenic virus but the sheer number of introduced mutations also makes it genetically extremely stable. Moreover, being a systematic method, it is seemingly applicable to several viruses. However, its suitability for attenuating large double-stranded DNA viruses like herpesviruses remained to be studied. Therefore, the present study dealt with the application of CPBD to attenuate a very virulent strain RB-1B of Marek’s disease virus (MDV), a highly contagious, lymphoproliferative and immunosuppressive herpesvirus that infects chickens. Due to its large size, CPBD of the entire MDV genome was impossible. Hence, meq, the major oncogene, was selected as a target gene. Despite being non-essential for viral growth in vitro, meq is a crucial immunogen as well as one of the few genes that are expressed during latency. CPB deoptimized meq showed lower protein production compared to the parental gene in vitro. A mutant virus, in which the parental meq was replaced with the CPB deoptimized version, showed comparable growth in cell culture, however, caused a slightly slower disease progression as well as decreased tumour incidence in vivo. These results indicate that although CPBD of the meq gene alone could not completely attenuate the virus, CPBD might be a suitable attenuation strategy for MDV.