RESUMO
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.
Assuntos
Códon/química , Gammaherpesvirinae/genética , Regulação Viral da Expressão Gênica , Engenharia Genética/métodos , Vírus Auxiliares/genética , Proteínas Imediatamente Precoces/genética , Transativadores/genética , Algoritmos , Animais , Pareamento de Bases , Linhagem Celular , Códon/metabolismo , Feminino , Fibroblastos/virologia , Gammaherpesvirinae/metabolismo , Células HEK293 , Vírus Auxiliares/metabolismo , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/prevenção & controle , Infecções por Herpesviridae/virologia , Vacinas contra Herpesvirus/biossíntese , Vacinas contra Herpesvirus/genética , Recombinação Homóloga , Humanos , Proteínas Imediatamente Precoces/imunologia , Camundongos , Camundongos SCID , Transativadores/imunologia , Replicação Viral/genéticaRESUMO
A tobacco mosaic virus (TMV)-based vector was utilized for expression of a cytosolic form of the bovine herpesvirus type 1 (BHV-1) protein glycoprotein D (gDc). Nicotiana benthamiana plants were harvested 7 days after inoculation with RNA transcripts derived from the TMV-gDc recombinant virus. Recombinant gDc protein of expected electrophoretic mobility accumulated in inoculated leaves to a concentration of about 20 micrograms/g of fresh leaf tissue. Oil-based vaccines were formulated with crude foliar extracts to immunize mice parentally. After a single injection, animals developed a sustained and specific response to both the isolated gD and native virus particles. Cattle vaccinated with the same gDc containing extracts developed specific humoral and cellular immune responses directed against both the viral gD and BHV-1 particles. Most importantly, animals vaccinated with the plant-produced gDc showed good levels of protection after challenge with the virulent BHV-1. Virus excretion was drastically reduced in these animals, reaching levels comparable to animals vaccinated with a commercial BHV-1 vaccine. The positive immunological characterization obtained for the gDc, indicated that an important part of the natural conformation was retained in the plant recombinant protein.
