HPV16-E6
expression in Saccharomyces cerevisiae: interaction with intrinsic proteins that
alter normal cell functioning.
Ana Pinto de Oliveira, Teresa Gonçalves
Microbiology, Faculty of Medicine, Rua Larga, Coimbra, 3004-504, Portugal
(apintooliveira@hotmail.com)
The malignant
phenotype of high-risk types human papillomavirus (HPV16 and HPV18) mainly
depends on the expression of two viral genes, E6 and E7. If control of E6 is
lost, this protein can trigger immortalization of cells and cancer. The aim of
this study is to determine if E6 protein interacts with yeast proteins that
alter the normal cell functioning, and the response to stress situations. In
the present work we expressed HPV16 E6 in S. cerevisiae and studied the response
to several stress situations. When incubated at 41°C, during 6 hours, an
E6-transformed strain presents higher cell viability than the wild type strain.
The expression of the E6 gene was assessed by mRNA quantification using RT-PCR
real time, and by Western blot protein analysis. The E6 expressing strain is
able to recover more efficiently from pheromone treatment. This same strain
exhibits growth in the presence of caffeine at a concentration of 20 mM, while
the wild type strain is only capable of growing until 10 mM. We conclude that
the heterologous expression of E6 in the yeast S. cerevisiae leads to an increased
resistance to stress conditions, showing that E6 interacts with the biochemical
machinery of yeast cells. A yeast two-hybrid system was used to study the
direct interactions between E6 and yeast proteins. For that a hybrid was
constructed using the strain PJ69-4A, with a plasmid containing our bait
protein (pGBDC.E6) and a plasmid with a yeast genomic library.