MECHANISM OF TRANSLATION OF THE BICISTRONIC MESSENGER-RNA ENCODING HUMAN PAPILLOMAVIRUS TYPE-16 E6-E7 GENES

The transforming genes E6 and E7 of human papillomavirus (HPV) type 16 and other HPV types are expressed from a bicistronic mRNA with a char acteristic spacing of 3 to 6 bp between the termination codon of E6 an d the initiation codon of E7. Plasmid pSP64E6E7 which contains the rea ding frames of both E6 and E7 was constructed in order to study the ex pression of both proteins in a coupled transcription/rabbit reticulocy te translation system. Both E6 and E7 proteins were expressed simultan eously. This translation could be interfered with by antisense oligonu cleotides corresponding to various regions of the transcript. Antisens e oligonucleotides targeted at sequences flanking either side of the t ranslation initiation codon of the E6 open reading frame were effectiv e in inhibiting the synthesis of both proteins, whereas oligonucleotid es complementary to the coding regions downstream of the first start c odon showed either a considerably reduced effect or none at all. In pa rticular, there was limited inhibition of E7 translation by antisense oligonucleotides flanking the translation start region of the E7 gene. In the presence of RNase H, it was possible to selectively inhibit th e synthesis of either E6 or E7 by several gene-internal antisense olig onucleotides. We conclude that HPV16 E6-E7 bicistronic mRNA is fully f unctional and that both proteins are translated with equal efficiency via the scanning mechanisms with reinitiation at the second open readi ng frame. In addition, both AE6 and AE7 may have therapeutical potenti al as they are capable of inhibiting the proliferation of CaSki cells which contain the HPV16 genome.