Expression of Foreign Proteins by Poliovirus Polyprotein Fusion: Analysis of Genetic Stability Reveals Rapid Deletions and Formation of Cardioviruslike Open Reading Frames

Abstract

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/3CDprocleavage 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.

Genetics, Pathogenesis and Evolution of Picornaviruses

“…Given the high frequency by which recombination occurs among sibling strands, a crossover mechanism may be favored, but decisive experiments to decide between these two mechanisms are lacking. In any case, a detailed study of genetic variations of polyprotein fusion vectors ( Figure 12.6D) strongly supports the model of parting and anchoring sites for template switching or loop-out deletion (Mueller and Wimmer, 1997;see below).…”

“…(+) FIGURE 12.7 Two models of illegitimate recombination during -RNA synthesis as was observed with an expression vector shown in Figure 12.6E (Mueller and Wimmer, 1998). Both models require a partial dissociation of the nascent-RNA from the template +RNA, caused presumably by pausing of the RNA polymerase.…”

“…Briefly, when expression vectors ( Figure 12.6E) consisting of a gag gene (encoding p17-p24; 1161 nt) of human immunodeficiency virus that was fused to the N-terminus of the poliovirus polyprotein (Andino et al, 1994;Mueller and Wimmer, 1998) were analysed after transfection into HeLa cells, the genomes were not only found to be severely impaired in viral replication but they were also genetically unstable (Mueller and Wimmer, 1997). Upon replication, the inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells.…”

“…Interestingly, the vector viruses did not readily revert to wt sequences but rather retained some of the insert plus the artificial 3C/3CD pr~ cleavage site (to allow processing at the N-terminus of the polyprotein). Thus, variants of different genotypes that replicated nearly as well as wt poliovirus had followed an evolutionary pathway towards the genetic organization of cardioviruses (Mueller and Wimmer, 1998). That is, the poliovirus polyprotein of these variants was preceded by gag-derived "leader" proteins of different but distinct sizes (predominantly between 20 and 50 aa long), the most prominent leader size reflecting the length of that in cardioviruses (67 aa).…”

“…That is, the poliovirus polyprotein of these variants was preceded by gag-derived "leader" proteins of different but distinct sizes (predominantly between 20 and 50 aa long), the most prominent leader size reflecting the length of that in cardioviruses (67 aa). In the immediate vicinity of the deletion borders of several isolates, short direct sequence repeats were observed that are likely to allow alignment of RNA strands for non-homologous (illegitimate) recombination during-strand synthesis (Figure 12.7;Mueller and Wimmer, 1998). Interestingly, the selection of the leader size occurred during the very first rounds of replication of the transfected RNA; in most cases, as sequential shortening of the leader sequence was not observed.…”