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 fusin g 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 poliovi rus polyprotein. All poliovirus expression vectors constructed by us a nd those obtained from Andino et al. were found to be severely impaire d in viral replication and genetically unstable. Upon replication, ins erted sequences were rapidly deleted as early as the first growth cycl e in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus th e artificial 3C(pro)/3CD(pro) cleavage site, engineered between the he terologous sequence and the poliovirus polyprotein, to give rise to ge notypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew b etter 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 rem aining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short dir ect sequence repeats that we propose are involved in aligning RNA stra nds for illegitimate (nonhomologous) RNA recombination during minus-st rand synthesis. On the basis of our results, which are at variance wit h published data, the utility of poliovirus vectors to express protein s >10 kDa in size through fusion with the polyprotein needs to be reev aluated.