Summary auto-generated
This study examined genetic changes in simian virus 40 (SV40) during tissue culture adaptation by analyzing two laboratory strains with detailed passage histories spanning over 25 years. Researchers sequenced the viral regulatory region and carboxy-terminal domain of the large T-antigen (T-ag) in low-passage stocks of SV40-Baylor and SV40-VA45-54 strains. Results showed that early passage stocks contained mixtures of viruses with either 'archetypal' regulatory regions (found in natural isolates, lacking enhancer duplications) or 'laboratory-type' regions (with duplicated 72 bp enhancer elements). Once the enhancer duplication was selected, the regulatory region structure remained genetically stable despite different passage histories. In contrast, the variable carboxy-terminal T-ag domain showed no sequence changes during tissue culture adaptation across multiple clones and passage lineages. Attempts to induce enhancer duplications by serially passaging archetypal SV40 strains in monkey cells for 14 passages were unsuccessful. Both archetypal and duplicated enhancer variants were viable in CV-1 cells, though duplicated versions produced larger plaques and higher titers. These findings suggest tissue culture adaptation involves either selection of rare pre-existing variants or extremely rare spontaneous duplication events.
Key findings
- Natural SV40 isolates contain 'archetypal' regulatory regions lacking enhancer duplications, while laboratory strains contain duplicated 72 bp enhancer elements; early passage stocks showed mixtures of both types
- Once a duplicated enhancer was selected during culture adaptation, the regulatory region structure remained genetically stable and did not revert, even after extensive passage
- The carboxy-terminal variable domain of T-ag showed no genetic drift during tissue culture adaptation across multiple cloned variants and passage lineages, suggesting this region is stable and could serve as a marker for strain classification
- Viruses with duplicated enhancers produced larger plaques and higher titers than archetypal strains in CV-1 and TC-7 cells, suggesting a growth advantage
- Serial passage of archetypal SV40 strains in permissive monkey cells for 14 passages did not induce enhancer duplications, indicating these changes are either extremely rare events or require conditions not present in laboratory cell culture
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Abstract
The regulatory region of natural isolates of simian virus 40 (SV40) is different from that of laboratory-adapted strains of the virus. The latter have a nucleotide sequence duplication within the enhancer region which varies slightly with each strain, whereas the duplication is lacking in fresh isolates of SV40, which contain an 'archetypal' regulatory region. Many isolates also display nucleotide differences in the DNA encoding the carboxy terminus of large tumour antigen (T-ag). To determine whether genetic changes in these two regions of the SV40 genome were detectable during laboratory adaptation and long-term passage, low-passage virus stocks of two laboratory strains which had detailed passage histories spanning more than 25 years (Baylor strain and VA45-54) were analysed using PCR, cloning and sequencing assays. Both laboratory and archetypal regulatory regions were present in low- passage stocks. Following duplication in the regulatory region, no additional changes were detectable. The variable region at the T-ag carboxy terminus did not undergo any change with tissue culture passage and may serve as a useful site for taxonomic classification of different strains of SV40. Cloned genomes containing single or duplicated enhancers derived from both SV40 strains were viable in CV-1 cells. Attempts to induce regulatory region duplications by 14 serial passages of SV40 archetypal strains in monkey cells were not successful. The results are compatible with tissue culture adaptation of SV40, reflecting either selection of a rare variant pre-existing in the original sample or generation of a rare regulatory region duplication in infected cells.