A point mutation in the DNA-binding domain of HPV-2 E2 protein has been found to increase its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter.
Researchers have used three forms of analytical research to discover the effects of point mutation in HPV-2 E2 protein. CAT expression assays, Western blots analysis and Electrophoresis mobility shift assays (EMSA) were utilised in the research, that found the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
CAT expression assays were used to find the enhanced promoter activity, indicating that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis then demonstrated that the transiently transfected E2 expressing plasmids were continuously expressed in the cells.
Finally, electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were far stronger than that of the HPV-2 prototype E2.
Posted by Fiona Griffiths