Cloning and sequencing of defective particles derived from the autonomous parvovirus minute virus of mice for the construction of vectors with minimal cis-acting sequences - PubMed (original) (raw)
Cloning and sequencing of defective particles derived from the autonomous parvovirus minute virus of mice for the construction of vectors with minimal cis-acting sequences
N Clément et al. J Virol. 2001 Feb.
Abstract
The production of wild-type-free stocks of recombinant parvovirus minute virus of mice [MVM(p)] is difficult due to the presence of homologous sequences in vector and helper genomes that cannot easily be eliminated from the overlapping coding sequences. We have therefore cloned and sequenced spontaneously occurring defective particles of MVM(p) with very small genomes to identify the minimal cis-acting sequences required for DNA amplification and virus production. One of them has lost all capsid-coding sequences but is still able to replicate in permissive cells when nonstructural proteins are provided in trans by a helper plasmid. Vectors derived from this particle produce stocks with no detectable wild-type MVM after cotransfection with new, matched, helper plasmids that present no homology downstream from the transgene.
Figures
FIG. 1
Different steps in the cloning of defective particles of MVM(p).
FIG. 2
Vectors derived from defective particles. The top line represents the WT MVM genome; only relevant restriction sites and MVM coordinates are indicated. Elements A and B (39) are represented as open boxes. Plasmids pULB3373 and pULB3377 (not depicted) are identical to pULB3375 and pULB3379, respectively, downstream from the IL-2 gene. They differ by the insertion site of IL-2 upstream from the transgene, which is the restriction site _Pfl_MI instead of _Hin_dIII.
FIG. 3
CsCl gradient fractionation of defective particles. Relevant fractions of one representative gradient are shown. They were revealed with an MVM probe on Southern blots. ssDNAs, single-stranded DNAs.
FIG. 4
Structure of defective particles. The top line indicates the WT MVM genome. Terminal palindromes (thin double lines) are not drawn to scale. The positions of the primers used for amplification of defective particles are indicated by arrows. Nucleotides are numbered according to the description of Astell et al. (1). Asterisks at the left mark inserts that were subcloned into pUC-MVM. Numbers between parentheses indicate the MVM coordinates of the start and the end of the deletion. Letters at the right of the deletion indicate single-base substitutions with their position. The sequence of the 65-bp deletion observed in two clones is given at the bottom of the figure.
FIG. 5
Sequence of deletion junctions. The sequences correspond to the plus strand of the genome (5′-to-3′ orientation). Coordinates of the internal deletion are listed on the right. The remaining copy of the short direct repeats is underlined. For pCR-D66 and pCR-D61, recombination involved repeats with one mismatched nucleotide; the sequence of the deleted copy is shown on top.
FIG. 6
Replication of defective particles. pCR-D plasmids (3 μg/6-cm-diameter dish), carrying defective particles, were cotransfected into NBE cells at a molar ratio of 1 with plasmid pULB3321, which provides NS1 protein in trans, allowing the excision and amplification of MVM DNA. Hirt's extracts prepared 2 days after transfection were separated by electrophoresis, blotted, and revealed with an MVM probe. The names of the defective particle clones are indicated above each lane. The lane marked with an asterisk shows a shorter exposure time from the same Southern blot. It contains an extract of cells transfected only with pULB3321.
FIG. 7
Replication of vectors derived from defective particles. The NS transcription unit of MVM(p) and the human IL-2 cDNA under the control of the P38 promoter were cloned into defective particles to generate vectors that were transfected into NBE cells (1 μg/6-cm-diameter dish). Control cells were transfected with pUC-MVM. The name of the transfected plasmid is indicated on top of each lane.
FIG. 8
IL-2 production after transfection of NBE cells with vectors derived from defective particles. Different vectors (3 μg/6-cm-diameter dish) were cotransfected with either of three helper plasmids (threefold molar excess), pSP116, pP38-VP, or pPSV40-VP. The first two helpers express capsid proteins under the control of the parvoviral P38 promoter, and the third uses the SV40 promoter. IL-2 levels were titrated 3 days after transfection in total lysates. Results of one representative experiment are shown.
FIG. 9
Transducing activity of vectors. Cells from the experiments described for Fig. 8 were lysed 3 days after transfection. The transducing activity of these lysates was measured as IL-2 expression of NBK cells, 3 days after infection. Results are from the same experiment as in Fig. 8.
FIG. 10
Transducing particles of recombinant and WT infectious virus were titrated by in situ hybridization with IL-2- and VP-specific probes, respectively. WT virus production is shown for two experiments that produced similar amounts of recombinant virus; results of the first experiment are shown in Fig. 8. The ratio of IL-2 produced after infection to transducing units was calculated for that experiment as well.
References
- Astell C R, Liu G, Brunstein J, Jindal H K, Tam P. Minute virus of mice cis-acting sequences required for genome replication and the role of the trans-acting viral protein NS1. Prog Nucleic Acids Res Mol Biol. 1996;55:245–285. - PubMed
- Besselsen D G, Pintel D J, Purdy G A, Besch-Williford C L, Franklin C L, Hook R R, Jr, Riley L K. Molecular characterization of newly recognized rodent parvoviruses. J Gen Virol. 1996;77:899–911. - PubMed
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