DNA-LAUNCHED INFECTIOUS CLONE WITH AN ARTIFICIAL INTRON: INFLUENCE ON VIRUS RESCUE AND GROWTH

Main Article Content

Authors

V.V. Keyer

National Center for Biotechnology, 13/5, Korgalzhyn road., Nur-Sultan, 010000, Kazakhstan

L.R. Syzdykova

National Center for Biotechnology, 13/5, Korgalzhyn road., Nur-Sultan, 010000, Kazakhstan

A.B. Shevtsov

National Center for Biotechnology, 13/5, Korgalzhyn road., Nur-Sultan, 010000, Kazakhstan

A.V. Shustov

National Center for Biotechnology, 13/5, Korgalzhyn road., Nur-Sultan, 010000, Kazakhstan

Abstract

A DNA-launched infectious clone is a plasmid that contains the full-genome cDNA copy of a viral genome under control of a eukaryotic promoter. Transfection of the plasmid in cell culture can then rescue the virus and allow for its growth. In this study, DNA-launched infectious clones of Venezuelan equine encephalitis virus (VEE) were produced. In the developed constructs, a cDNA copy of the VEE genome was placed under control of the cytomegalovirus (CMV) promoter, and a ribozyme and polyadenylation signal were engineered at the 3′-end of the sequence. Moreover, a small hybrid intron (composed from parts of the second beta-globin intron and the IgG intron) was cloned into the junction between the viral 5′-untranslated region (UTR) and the nsP2 gene, and the effects of inserting an intron in the DNA-launched infectious clone on the rescue efficiency and growth kinetics were assessed.The rescue efficiency was high for all constructs at 4.8 × 104 focus-forming units (FFU)/ mg of transfected plasmid DNA for the parental construct, 4.0 × 104 FFU/mg for the construct with a non-natural PstI site between the 5′UTR and nsP2 gene, and 1.0 × 104 FFU/mg for the construct with the intron placed in a selected genomic position. The three rescued viruses reached similar titers, indicating that the intron does not have a major effect on the rescuing efficiency. Thus, we have demonstrated an efficient method of cloning introns into natural or engineered PstI sites to achieve efficient viral rescue and growth.

Keywords

alphavirus, Venezuelan equine encephalitis virus, DNA-launched infectious clone, intron, virus rescue, CMV promoter

Article Details

References

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