Full-length wild-type SV40 VP1 and its derivative mutants were constructed with the pGEX-4T2 plasmid (GE Healthcare, Piscataway, NJ), expressed in Escherichia coli BL21(DE3) cells (Agilent) and purified by using glutathione S-transferase (GST) affinity purification and thrombin cleavage, as previously described (21 (link)). Mutant VP1 unable to form VLPs was truncated after amino acid position 305 (21 (link)).
For electron microscopy, purified pentamers were diluted in pH 7.2 PBS at 20 µg/ml and analyzed after negative staining with 1% uranyl acetate (21 (link)) via Tecnai T12 transmission electron microscopy at the Yale Electron Microscopy Core Facility. For transmission electron microscopy of infected cells, 2 × 106 CV-1 cells were mock infected or infected with wild-type or A70L SV40 at an MOI of 1.5. At 64 h postinfection, samples were fixed, stained with 2% uranyl acetate, dehydrated, embedded, sectioned, and mounted onto copper grids as described elsewhere (40 (link)).
Purified pentamers were added to CV-1 cells maintained in DMEM containing 1% (vol/vol) FBS.
For electron microscopy, purified pentamers were diluted in pH 7.2 PBS at 20 µg/ml and analyzed after negative staining with 1% uranyl acetate (21 (link)) via Tecnai T12 transmission electron microscopy at the Yale Electron Microscopy Core Facility. For transmission electron microscopy of infected cells, 2 × 106 CV-1 cells were mock infected or infected with wild-type or A70L SV40 at an MOI of 1.5. At 64 h postinfection, samples were fixed, stained with 2% uranyl acetate, dehydrated, embedded, sectioned, and mounted onto copper grids as described elsewhere (40 (link)).
Purified pentamers were added to CV-1 cells maintained in DMEM containing 1% (vol/vol) FBS.
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