Efficient intracellular assembly of papillomaviral vectors.
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.
Buck
Christopher B
CB
Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, Maryland 20892-4263, USA.
Pastrana
Diana V
DV
Lowy
Douglas R
DR
Schiller
John T
JT
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D052688:Bovine papillomavirus 1 / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D010957:Plasmids; D017422:Sequence Analysis, DNA; D014162:Transfection; D019065:Virus Assembly
D000914:Antibodies, Viral; D036022:Capsid Proteins; C085217:L1 protein, Bovine papillomavirus; C085218:L2 protein, Bovine papillomavirus; C063316:L2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; C063317:L1 protein, Human papillomavirus type 16