Figure 1
From: SV40-Immortalized Human Fibroblasts as a Source of SV40 Infectious Virions
Complete molecular characterization of the SV40 particles present in the MRC5-SV2 immortalized human fibroblast cell line, from the DNA to the protein level. A: PCR amplification with RA1 and RA2 primers (Lednicky and Butel 2001[40]) of the SV40 regulatory region. 45-54-2 viral DNA was extracted from the culture medium of MRC5-SV2 cells as described in Materials and Methods. The DNA of SV40 strain 776, used as control, shows a single PCR product of 314 bp (arrow), as expected. In SV40 strain 45-54-2 the RA1 primer anneals on 2 repeated sequences, with the consequent amplification of 2 fragments, one of 335 bp and the other of 242 bp (arrows). R-, control of the PCR without DNA template; M, 100 base-pair ladder (Amersham, Milano, Italy) as marker. B, C: RT-PCR experiments on cDNA from MRC5-SV2 cells. A single reverse transcription reaction was performed on MRC5-SV2 total cytoplasmic RNA extracted from 106 cells using the Rneasy kit (Qiagen, Milano, Italy). Genomic DNA extracted from MRC5-SV2 cells was amplified for comparison. SV40 DNA, strain 776, was employed as positive control, whereas normal human DNA was used as negative control. R−, control of the PCR without DNA template; M, molecular weight marker VI (Roche Diagnostics, Monza, MI, Italy). B: Amplification carried out using a primer pair located upstream and downstream the Tag intron, which generates an amplimer of 229 bp on the cDNA and one of 575 on the genomic DNA (arrows) (26). C: Amplification of 409 bp within the VP1 sequence obtained with the primer pair VPA-VPB (Martini et al., 2002 (26)). (D) Western blot analysis carried out on total protein extracts from 105 cells separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A goat anti-SV40 serum was used for primary incubation, followed by an anti-goat IgG coupled to peroxidase for detection. Protein extract form CV-1 cells harvested 24 h after SV40 infection, was used as positive control, whereas protein extract from normal human MRC5 fibroblasts was the negative control.