Structures of standard MVMp- and hH1-based vectors, derived chimeras, and helper plasmids. (A) The standard parvoviral vectors were derived from the modified infectious DNA clones (pMVMp and phH1) by deleting about 800 bp from the coding sequence of the structural VP1 and VP2 capsid genes and inserting a MCS (white box) at the VP2 translation initiation codon. Both promoters (P4 and P38) are indicated by arrows. The region encoding the nonstructural proteins (NS1/NS2) is shown. Chimeric MVMp-based vector DNA was constructed by replacing a BamHI-BglI fragment from pMVMpΔ800 with the corresponding region of phH1Δ800, resulting in pChi-MVMpΔ800. Chimeric hH1-based vector DNA was obtained by inserting the BamHI-BglI fragment of MVMpΔ800 into phH1Δ800 cleaved with the same restriction enzymes, resulting in pChi-hH1Δ800. The MVMp (black boxes) and hH1 (grey boxes) DNA sequences are indicated. GFP or EGFP was cloned in standard (pMVMp/GFP and phH1/GFP) and chimeric (pChi-MVMp/EGFP and pChi-hH1/EGFP) DNA vectors by inserting a 731-bp NotI restriction fragment from plasmid pTR/UF2 (30) or a 784-bp SacI-NotI restriction fragment from plasmid pEGFP-1 (Clontech), respectively, into the vectors digested with the same restriction enzymes. (B) DNA helper constructs. The VP1 and VP2 genes from either MVMp or H-1 virus are expressed under the control of the immediate-early promoter of human CMV (P-CMV). Recombinant parvoviruses were produced by cotransfection of human 293T cells with the vector DNA and helper plasmid as indicated in the text. SV40, simian virus 40.

Contamination of recombinant virus stocks by RCVs. Standard and chimeric recombinant virus stocks were produced by cotransfecting 293T cells with vector DNA and the corresponding helper plasmid (described in the legend of Fig. 1), while pseudotype virus stocks were produced by cotransfection of standard pMVMp/GFP and phH1/GFP with H1 helper and MVMp helper DNA, respectively, resulting in MVMp/GFP(H1) and hH1/EGFP(MVM). Virus stocks were harvested 72 h posttransfection and purified by centrifugation on iodixanol gradients, and virus titers were determined on NB324K cells by a hybridization assay using a specific ³²P-labeled NS DNA probe consisting of a 703-bp EcoRV-EcoRI restriction fragment of either pMVMΔ800 or phH1Δ800. Total levels of virus production are expressed as RU per 2 × 10⁶ transfected cells (grey bars). The proportion of replication-competent viruses present in the recombinant virus stocks was determined by plaque assay and expressed as PFU per 2 × 10⁶ transfected cells (black bars). The results shown are the averages of at least three independent experiments, with standard deviations indicated by the error bars.

Viral DNA amplification after infection of human and mouse cells with chimeric and pseudotyped viruses. (A and B) NB-324K and A9 cells were infected at a MOI of 0.001 RU/cell (A) or 0.03 RU/cell (B) (MVMp-based virus) and 3 RU/cell (B) (hH1-based virus) with the indicated virus stocks and transferred to nitrocellulose membranes 48 h postinfection. Titration of recombinant viruses was performed by hybridization assay and autoradiography (16, 17), using a NS-specific DNA probe as described in the legend to Fig. 2. (C) Southern blotting analysis of low-molecular-weight DNA, isolated by the modified Hirt procedure, 72 h after infection of A9 cells with the indicated virus stocks at a MOI of 3 RU/cell. DNA samples were fractionated by agarose gel electrophoresis, transferred to Hybond N+ nylon membrane, and hybridized with a NS-specific DNA probe. The positions of monomer- and dimer-length RFs are indicated.

Expression of NS1 and NS2 proteins in A9 and NB-324K cells. (A) Cells were mock treated or infected at a MOI of 5 PFU/cell with wild-type MVMp (MVMp-wt) or H-1 (H1-wt) virus. At 18 h postinfection, cell cultures were metabolically labeled for 2 h with 200 μCi of Trans³⁵S label (1,000 Ci/mmol; ICN Pharmaceuticals) in Met- and Cys-free Eagle's minimum essential medium supplemented with 5% dialyzed fetal calf serum. Cells were then lysed in modified radioimmunoprecipitation assay (MRIPA) buffer (10 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate) containing a mixture of protease inhibitors (Complete; Roche Molecular Biochemicals). Equal amounts of labeled protein extracts (10⁷ cpm) were immunoprecipitated with 10 μl of the polyclonal rabbit antiserum SP11 directed against the N terminus of NS1 and NS2 proteins of MVMp virus (3). The immunocomplexes were fractionated by electrophoresis on a sodium dodecyl sulfate-8% polyacrylamide gel and revealed by autoradiography. (B) A9 cells were transfected with 4 μg of plasmid DNA using the Lipofectamine transfection reagent as recommended by the supplier (Gibco BRL), while 25 μg of plasmid DNA was used to transfect NB-324K cells by the calcium phosphate precipitation method. Transfection efficiency was determined by the percentage of (E)GFP-positive cells and was identical for all the constructs. At 48 h after transfection, cells were lysed in MRIPA buffer, and 50-μg portions of protein extracts were analyzed by Western blotting. NS1 and NS2 were revealed by incubating the membrane with rabbit polyclonal antisera directed against the C-terminal part of MVMp NS1 (SP7) (13) and against the major isoform of MVMp NS2 (NS2p), respectively. The NS2p-specific antiserum was producedusing the keyhole limpet hemocyanin-coupled peptide AELGLRPEITWF. The membrane was further incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody, and the immunoreactive proteins were visualized using Western Lightning Chemiluminescence Detection Reagent Plus (Perkin-Elmer Life Sciences).

Cell tropism of chimeric and pseudotyped virus MVMp and hH1 vectors. Several human (A) and mouse (B) cell lines were infected with recombinant standard, chimeric, or pseudotyped virus stocks at a MOI of 3 or 10 RU/cell, respectively. GFP (or EGFP) expression was monitored with a fluorescence microscope 48 and 72 h postinfection. The line with the highest number of fluorescent cells after infection with the indicated virus stock was set at 100% for GFP-positive cells. GFP-positive cells from the same cell line infected with all the other virus stocks were expressed relative to this reference value (given as a percentage). A minimum of 200 GFP-positive cells were counted in each cell line. Results are of one representative experiment (of three). (A) Relative number of GFP-positive human cells (U937 , U373 , and HeLa ) after infection with the indicated recombinant virus stocks. (B) Relative numbers of GFP-positive mouse cells (A9 , L929 , Ehrlich ascitic fluid , and C3H10T1/2 ) after infection with the indicated recombinant virus stocks.