title: A codon-shuffling method to prevent reversion during production of replication-defective herpesvirus stocks: Implications for herpesvirus vaccines.
abstract: Herpesviruses establish life-long chronic infections that place infected hosts at risk for severe disease. Herpesvirus genomes readily undergo homologous recombination (HR) during productive replication, often leading to wild-type (WT) reversion during complementation of replication-defective and attenuated viruses via HR with the helper gene provided in trans. To overcome this barrier, we developed a synthetic-biology approach based on a technique known as codon shuffling. Computer-assisted algorithms redistribute codons in a helper gene, thereby eliminating regions of homology, while enabling manipulation of factors such as codon-pair bias and CpG content to effectively titrate helper-gene protein levels. We apply this technique to rescue the replication of a murine gammaherpesvirus engineered with a mutation in the major immediate-early transactivator protein RTA. Complementation with codon-shuffled RTA constructs did not yield any WT revertant virus, a sharp contrast to WT virus contamination frequently observed during complementation with an unmodified helper gene. We further demonstrate the importance of eliminating WT virus contamination in an animal model of gammaherpesvirus lethality. We propose complementation by codon shuffling as a means to produce replication-defective or attenuated viruses. This method has immediate utility for investigating roles of essential genes in viral replication and will better enable future development of herpesvirus vaccines.
authors.affiliation: Department of Microbiology and Immunology and Center for Microbial Pathogenesis and Host Inflammatory Responses, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Computer Science, Stony Brook University, Stony Brook,New York, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Ward
authors.affiliation: Department of Computer Science, Stony Brook University, Stony Brook,New York, USA.
authors.forename: Rukhsana
authors.initials: R
authors.lastname: Yeasmin
authors.affiliation: Department of Computer Science, Stony Brook University, Stony Brook,New York, USA.
authors.forename: Steven
authors.initials: S
authors.lastname: Skiena
authors.affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Laurie T
authors.initials: LT
authors.lastname: Krug
authors.affiliation: Department of Microbiology and Immunology and Center for Microbial Pathogenesis and Host Inflammatory Responses, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors.forename: J Craig
authors.initials: JC
authors.lastname: Forrest
mesh_terms: D000465:Algorithms; D000818:Animals; D020029:Base Pairing; D002460:Cell Line; D003062:Codon / Q000737:chemistry* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000821:virology; D018142:Gammaherpesvirinae / Q000235:genetics* / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006378:Helper Viruses / Q000235:genetics* / Q000378:metabolism; D006566:Herpesviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D022283:Herpesvirus Vaccines / Q000096:biosynthesis / Q000235:genetics; D059765:Homologous Recombination; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016513:Mice, SCID; D015534:Trans-Activators / Q000235:genetics* / Q000276:immunology; D014779:Virus Replication / Q000235:genetics
chemical_list: D003062:Codon; D022283:Herpesvirus Vaccines; D017874:Immediate-Early Proteins; C417604:Rta protein, Human herpesvirus 8; D015534:Trans-Activators