HPV16 PsVs were prepared as previously described63 (link). In brief, expression plasmids carrying codon-optimized L1 and L264 (link) expression vector pShell 16L1/L2wt65 (link) were co-transfected with a pcDNA3.1 luciferase reporter plasmid66 (link) into HEK 293TT cells using polyethylenimine. For PsVs used in infection assays, the pcDNA3.1 luciferase reporter plasmid was replaced by the promoter-reporter plasmid pGL4.20 containing the HPV16 long control region (LCR) and the HPV16 early promoter regulating the luciferase expression as described earlier67 (link),68 (link). For detection of the DNA, EdU-modified PsVs were used. After transfection of pShell 16L1/L2wt and pcDNA3.1 plasmids the cell culture medium was supplemented with 20 μM 5-ethynyl-2′-deoxyuridine (EdU, Click-iT AlexaFluor® 488 Imaging Kit, Thermo Fisher Scientific), to enable the staining of the DNA.
48 hours after transfection, cells were lysed and the pseudoviruses were purified by gradient centrifugation using OptiPrep (Sigma-Aldrich). Quantification of the pseudovirions (viral genome equivalents (vge)) was performed by quantitative PCR using a 7500 Real-Time PCR System and Sequence Detection Software v2.3 (Applied Biosystems, Foster City, CA, USA)69 (link).
48 hours after transfection, cells were lysed and the pseudoviruses were purified by gradient centrifugation using OptiPrep (Sigma-Aldrich). Quantification of the pseudovirions (viral genome equivalents (vge)) was performed by quantitative PCR using a 7500 Real-Time PCR System and Sequence Detection Software v2.3 (Applied Biosystems, Foster City, CA, USA)69 (link).
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