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. 1998 Jan;72(1):20-31.
doi: 10.1128/JVI.72.1.20-31.1998.

Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames

S Mueller  1 , E Wimmer
Affiliations

Expression of foreign proteins by poliovirus polyprotein fusion: analysis of genetic stability reveals rapid deletions and formation of cardioviruslike open reading frames

S Mueller et al. J Virol. 1998 Jan.

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/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.

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Figures

FIG. 1
FIG. 1
(A) Genomic organization of expression constructs. Amino acids in capital letters are part of the PV genome; lowercase amino acids mark exogenous sequences, with boxes indicating the coding sequences of the inserted genes. The PV ORF initiating AUG is in its original context; the first 3 aa of VP4 (GAQ) are duplicated. The differences in pmogfp and pmoHgag are a more extensive multiple cloning site, six glycine residues upstream the cleavage site instead of four, and inclusion of the original P5 residue (glutamate) into the 3Cpro/3CDpro recognition site. (B) Schematic of the oligonucleotides used as RT-PCR primers and resulting fragment lengths.
FIG. 2
FIG. 2
Plaque phenotypes of GFP- and Gag-expressing constructs. Virus isolated from RNA transfections and after the fifth passage were plaque assayed on 35-mm-diameter six-well plates of HeLa R19 monolayers (considered first and sixth passages, respectively). After 48 h of incubation at 37°C and 5% CO2, the cells were stained with crystal violet.
FIG. 3
FIG. 3
RT-PCR analysis of PVMgfp2 (A), mogfp (B), PVMgag2 (C), and moHgag (D) virus variants with oligonucleotides flanking the exogenous sequences [7215, nt 667 to 690 of PV1(M); 6509, nt 851 to 877 of PV1(M)]. Two independent RNA transfections for each construct were done (A and B). Reinfection for RNA purification was considered first passage (1.). Each transfection was passaged five more times in duplicates (6.). Deletions within the foreign sequences result in shorter bands, accordingly. M, DNA molecular weight marker VI (Boehringer Mannheim); P, PCR product of the respective plasmid; T, RT-PCR of the respective transcript RNA. Products were analyzed on a 1.2% agarose gel. Sizes are indicated in nucleotides.
FIG. 4
FIG. 4
Genetic stability of deletion variants of plaque-purified (p.p.) moHgag. Virus recovered from RNA transfections A and B was plaque assayed. Two small plaques from each plate were picked (A1, A2, B1, and B2), grown up (1.), and passaged four more times (5.). Total RNA was subjected to RT-PCR with primers 7215 and 6509. M, DNA molecular weight marker VI (Boehringer Mannheim); T, RT-PCR of pmoHgag transcript RNA; wt, RT-PCR of PV1(M) virion RNA. Products were analyzed on a 1.2% agarose gel. Sizes are indicated in nucleotides.
FIG. 5
FIG. 5
Competition in RT-PCR. Transcript RNAs of pT7PVMgag2 and pT7PVM (wt) were mixed as indicated to a total of 200 ng and subjected to RT-PCR with primers 7215 and 6509. M, DNA molecular weight marker VI (Boehringer Mannheim). Products were run on a 1.2% agarose gel (ethidium bromide stained).
FIG. 6
FIG. 6
Selective RT-PCR using one flanking primer [01, nt 487 to 508 of PV1(M)] and one primer mapping within the inserted sequence (7404, nt 309 to 331 of gfp10 ORF [A], or 7403, nt 286 to 309 of HIV-1 gag [B]). M, DNA molecular weight marker VI (Boehringer Mannheim); T, RT-PCR of the respective transcript RNA; 1., first passage; 6., sixth passage; wt, RT-PCR of PV1(M) virion RNA (negative control; neither 7404 nor 7403 can anneal). Products were analyzed on a 1.2% agarose gel (ethidium bromide stained). Sizes are indicated in nucleotides.
FIG. 7
FIG. 7
Determination of the ratio of moHgag deletion variants during progressive passaging. Viruses were plaque purified from initial RNA transfection (A), first passages (A and B), and sixth passages (C). Viral RNAs were subjected to RT-PCR with flanking primers 7215 and 6509. M, DNA molecular weight marker VI (Boehringer Mannheim); T, RT-PCR of pmoHgag transcript RNA; wt, RT-PCR of PV1(M) virion RNA. Products were run on a 1.2% agarose gel (ethidium bromide stained).
FIG. 8
FIG. 8
Sequence analysis of moHgag and PVMgag2 variants. Variant genotypes were divided into six and three categories based on the size of the retained foreign sequence as determined by RT-PCR (see text) (a, b, c, d, e, and f for moHgag; β, γ, and δ for PVMgag2). Several variants of each category were sequenced by cycle sequencing of their RT-PCR products. Black boxes represent gag-specific sequences. Dotted lines mark deleted portions (drawn to scale). Asterisks and dots indicate the 5′ and 3′ borders, respectively, of each deletion. Numerals above the deletions identify positions in the parental plasmid (pmoHgag or pT7PVMgag2) of the last upstream nucleotide before and the first downstream nucleotide after each deletion. The following deviations from the sequence shown were found in plasmid pmoHgag: position 1872, A→G (Glu→Gly); position 1875, G→T (Gly→Val) (both map within the linker/cleavage site and are also present in mogfp, which originates from the same vector plasmid); and position 1184 A→G (Ile→Val).
FIG. 9
FIG. 9
Sequence alignments of the regions surrounding the deletion borders in moHgag (A) and PVMgag2 (B) variants. Thirty nucleotides around the 5′ and 3′ borders of each deletion (15 nt upstream and downstream) were aligned and analyzed for homologies. The upper sequence shows the 5′ border, and the lower sequence shows the 3′ border, with asterisks and dots indicating the respective deletion endpoints (see also Fig. 8 for positions of these marks). Note that both sequences are positive sense and can be located either on the same template strand or on two sibling strands. Numerals refer to positions within the full-length cDNA clones, pmoHgag and pT7PVMgag2. Capital letters represent the sequence of the variant (upper left to lower right) as determined by sequencing; lowercase letters show deleted parental sequences. Sequence homologies are boxed.
FIG. 10
FIG. 10
Two possible models of illegitimate recombination during minus-strand synthesis as exemplified for variant moHgag c1. Both models require a partial dissociation of the nascent minus [(−)] strand from the template plus [(+)] strand, caused by pausing of the polymerase. The free 3′ end of the nascent strand can reanneal to a short complementary sequence further upstream on the same template strand, thereby looping out the intervening sequences (A), or can reanneal to the same complementary sequence, but on a sibling plus strand, and complete synthesis on this second template (strand switching [B]). In both cases, the resulting minus strands would have excised the sequence between nt 893 and 1798 and can now, in turn, give rise to truncated positive-sense RNA genomes.
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