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Journal of Virology, October 2009, p. 9957-9969, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.00508-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic Inactivation of Poliovirus Infectivity by Increasing the Frequencies of CpG and UpA Dinucleotides within and across Synonymous Capsid Region Codons{triangledown} ,{dagger}

Cara C. Burns,* Ray Campagnoli, Jing Shaw, Annelet Vincent, Jaume Jorba, and Olen Kew

Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333

Received 11 March 2009/ Accepted 9 July 2009

Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only ~9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5°C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.

* Corresponding author. Mailing address: Polio and Picornavirus Branch, G-10, Division of Viral Diseases, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-5499. Fax: (404) 639-4011. E-mail: CBurns{at}cdc.gov

{triangledown} Published ahead of print on 15 July 2009.

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/.

Journal of Virology, October 2009, p. 9957-9969, Vol. 83, No. 19
0022-538X/09/$08.00+0     doi:10.1128/JVI.00508-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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