Efficient intracellular assembly of papillomaviral vectors

Abstract

Although the papillomavirus structural proteins, L1 and L2, can spontaneously coassemble to form virus-like particles, currently available methods for production of L1/L2 particles capable of transducing reporter plasmids into mammalian cells are technically demanding and relatively low-yield. In this report, we describe a simple 293 cell transfection method for efficient intracellular production of papillomaviral-based gene transfer vectors carrying reporter plasmids. Using bovine papillomavirus type 1 (BPV1) and human papillomavirus type 16 as model papillomaviruses, we have developed a system for producing papillomaviral vector stocks with titers of several billion transducing units per milliliter. Production of these vectors requires both L1 and L2, and transduction can be prevented by papillomavirus-neutralizing antibodies. The stocks can be purified by an iodixanol (OptiPrep) gradient centrifugation procedure that is substantially more effective than standard cesium chloride gradient purification. Although earlier data had suggested a potential role for the viral early protein E2, we found that E2 protein expression did not enhance the intracellular production of BPV1 vectors. It was also possible to encapsidate reporter plasmids devoid of BPV1 DNA sequences. BPV1 vector production efficiency was significantly influenced by the size of the target plasmid being packaged. Use of 6-kb target plasmids resulted in BPV1 vector yields that were higher than those with target plasmids closer to the native 7.9-kb size of papillomavirus genomes. The results suggest that the intracellular assembly of papillomavirus structural proteins around heterologous reporter plasmids is surprisingly promiscuous and may be driven primarily by a size discrimination mechanism.

FIG. 1.

Expression of E1 and E2 proteins fails to enhance BPV1 vector production efficiency. 293H target cells were incubated for 48 h with various types of producer cell lysates. Ten thousand target cells were analyzed by FACS to detect GFP expression resulting from BPV1 vector transduction. Producer cells were generated by cotransfection of 293H cells with an L1/L2 expression plasmid (pSheLL) and the target plasmid shown, together with either control plasmid pCAT (striped bars) or pE1/E2 (solid bars). Target plasmid names denote the presence or absence of the BPV1 URR (U) or of a putative packaging signal (P) and the plasmid size (in base pairs). Two negative controls for BPV1 vector transduction are shown at left.

FIG. 2.

Target plasmid size influences BPV1 vector production efficiency. The graph shows the number of 293H target cells expressing GFP after treatment with cell lysates generated by cotransfection of pSheLL together with GFP-expressing plasmids of various sizes. See the legend to Fig. 1 for a description of target plasmid nomenclature.

FIG. 3.

Quantitation of low-molecular-weight and nuclease-resistant DNA in transiently transfected 293TT cells. The top image shows a SYBR Green I-stained agarose gel loaded with low-molecular-weight DNA extracted from 293TT cells transfected with various plasmids. Cells were cotransfected with the following: lane 1, pU-10151 and pSU-5697; lane 2, pADAP-L1P and pSU-5697; lane 3, pSheLL and pSU-5697; lane 4, pU-10151 and pU-5864; lane 5, pSheLL and pU-5864. In the top image, either pSheLL or a control plasmid (pADAP-L1P or pU-10151) is visible above the band marked “target.” Each lane in the top image was loaded with 100,000 cell equivalents of low-molecular-weight DNA linearized by digestion with the restriction enzyme BsrGI. The lower image shows nuclease-resistant DNA isolated from same transfectants. The nuclease-resistant DNA was linearized by digestion with BsrGI and visualized by SYBR Green I staining of an agarose gel. Each lane of the gel in the bottom image was loaded with 1.5 million cell equivalents of extracted DNA. The chart at the bottom of the figure shows the presence or absence of various components within the producer cells and the BPV1 vector titer of the cell lysate.

FIG. 4.

Purification of a BPV1 vector stock produced by transfection of 293TT cells. The top image depicts a protein-stained polyacrylamide gel loaded with samples from various fractions of an OptiPrep gradient. Western blotting of the fractions using an anti-L1 antibody showed a pattern identical to the band marked as L1 and showed negligible amounts of L1 above fraction 11 (data not shown). Lane m contains protein standards marked in kilodaltons (top image) or DNA standards marked in kilobases (lower image). The lower image shows nuclease-resistant DNA precipitated from each OptiPrep fraction and visualized by agarose gel electrophoresis followed by SYBR Green I staining. The target plasmid DNA migrated at the same rate as the supercoiled target plasmid standard. The BPV1 vector titer of each cell lysate (in transducing units per milliliter) is given in the chart at the bottom of the figure.