Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames.
Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448-1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDpro cleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins > 10 kDa in size through fusion with the polyprotein needs to be reevaluated.
Mueller
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Department of Molecular Genetics and Microbiology, School of Medicine, State University of New York at Stony Brook, 11794, USA. mueller@asterix.bio.sunysb.edu
Wimmer
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E
D000595:Amino Acid Sequence; D001483:Base Sequence; D017943:Cardiovirus / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics; D014644:Genetic Variation; D005822:Genetic Vectors; D005838:Genotype; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017955:Poliovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D011995:Recombination, Genetic; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014614:Vaccines, Synthetic / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics*
D017931:DNA Primers; D015683:Gene Products, gag; D008164:Luminescent Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D014764:Viral Proteins; D049452:Green Fluorescent Proteins