1. Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.
- Author
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Song Y, Gorbatsevych O, Liu Y, Mugavero J, Shen SH, Ward CB, Asare E, Jiang P, Paul AV, Mueller S, and Wimmer E
- Subjects
- A549 Cells, HeLa Cells, Humans, Phenotype, Poliomyelitis genetics, RNA, Viral, Genome, Viral, Poliomyelitis virology, Poliovirus genetics, Poliovirus pathogenicity, Virion genetics, Virus Assembly, Virus Replication
- Abstract
Computer design and chemical synthesis generated viable variants of poliovirus type 1 (PV1), whose ORF (6,189 nucleotides) carried up to 1,297 "Max" mutations (excess of overrepresented synonymous codon pairs) or up to 2,104 "SD" mutations (randomly scrambled synonymous codons). "Min" variants (excess of underrepresented synonymous codon pairs) are nonviable except for P2
Min , a variant temperature-sensitive at 33 and 39.5 °C. Compared with WT PV1, P2Min displayed a vastly reduced specific infectivity (si) (WT, 1 PFU/118 particles vs. P2Min , 1 PFU/35,000 particles), a phenotype that will be discussed broadly. Si of haploid PV presents cellular infectivity of a single genotype. We performed a comprehensive analysis of sequence and structures of the PV genome to determine if evolutionary conserved cis-acting packaging signal(s) were preserved after recoding. We showed that conserved synonymous sites and/or local secondary structures that might play a role in determining packaging specificity do not survive codon pair recoding. This makes it unlikely that numerous "cryptic, sequence-degenerate, dispersed RNA packaging signals mapping along the entire viral genome" [Patel N, et al. (2017) Nat Microbiol 2:17098] play the critical role in poliovirus packaging specificity. Considering all available evidence, we propose a two-step assembly strategy for +ssRNA viruses: step I, acquisition of packaging specificity, either ( a ) by specific recognition between capsid protein(s) and replication proteins (poliovirus), or ( b ) by the high affinity interaction of a single RNA packaging signal (PS) with capsid protein(s) (most +ssRNA viruses so far studied); step II, cocondensation of genome/capsid precursors in which an array of hairpin structures plays a role in virion formation., Competing Interests: Conflict of interest statement: S.M. and E.W. are co-founders of Codagenix, Inc., a small biotech company focusing on the design and development of vaccines. They are co-holders of a patent describing the effects of changes of synonymous codon pairs in viral genomes. S.M is an employee of Codagenix. The work described in this manuscript presents no conflict of interest with the focus of Codagenix. A.V.P. has retired with no connection to Codagenix. Y.L., S.C., and C.B.W. have left the laboratory >2 years ago; their employments are unrelated to Codagenix. P.J. is currently unemployed. Y.S., A.G., J.M., E.A. are laboratory members with no connection to Codagenix.- Published
- 2017
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