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Mutational analysis of open reading frame E4 of bovine papillomavirus type 1
Abstract:
Open reading frame (ORF) E4 is a 353-base-pair ORF of bovine papillomavirus type 1. To determine the biological activities of this ORF in mouse C127 cells, we analyzed the effects of two constructed mutations which are predicted to prevent synthesis of ORF E4 proteins while leaving the amino acid sequence encoded by the overlapping ORF E2 unchanged. Neither mutation interfered with the abilities of the mutants to efficiently induce focus formation, induce growth in soft agarose, or transactivate an inducible bovine papillomavirus type 1 enhancer. Also, neither mutation prevented establishment of the viral DNA as an extrachromosomal plasmid in transformed cells. These results suggest that ORF E4 proteins are not required for these biological activities, and they are consistent with the observation of others (J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986) that the ORF E4 protein of a human papillomavirus is associated with late gene expression during papilloma formation.
Insights
Bovine papillomavirus type 1 open reading frame (ORF) E4 proteins are not essential for key cellular transformation activities. Mutations in ORF E4 did not affect focus formation, anchorage-independent growth, or viral enhancer activation in mouse cells.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Bovine papillomavirus type 1 (BPV-1) is a virus known to cause warts in cattle.
- Open reading frames (ORFs) encode proteins crucial for viral replication and pathogenesis.
- ORF E4's specific role in BPV-1 infection and transformation has been unclear.
Purpose of the Study:
- To investigate the biological functions of the bovine papillomavirus type 1 (BPV-1) ORF E4 in mouse C127 cells.
- To determine if ORF E4 proteins are necessary for viral transformation and replication-related activities.
Main Methods:
- Constructed two mutations in BPV-1 ORF E4 predicted to abolish protein synthesis.
- Assessed the impact of these mutations on focus formation, growth in soft agarose, and enhancer transactivation in mouse C127 cells.
- Examined the maintenance of viral DNA as extrachromosomal plasmids in transformed cells.
Main Results:
- Mutations preventing ORF E4 protein synthesis did not impair focus formation.
- Mutant viruses efficiently grew in soft agarose, indicating no requirement for ORF E4 in anchorage-independent growth.
- Transactivation of the BPV-1 enhancer by the viral E2 protein remained unaffected by ORF E4 mutations.
- Viral DNA was successfully established as extrachromosomal plasmids in cells transformed by the mutant viruses.
Conclusions:
- BPV-1 ORF E4 proteins are dispensable for critical biological activities including transformation and viral DNA maintenance.
- These findings align with observations linking human papillomavirus ORF E4 proteins to late-stage gene expression during papilloma formation.
- The study clarifies the non-essential role of BPV-1 ORF E4 in early viral transformation events.