SINGLE AMINO-ACID SUBSTITUTIONS IN LOW-RISK HUMAN PAPILLOMAVIRUS (HPV) TYPE-6 E7 PROTEIN ENHANCE FEATURES CHARACTERISTIC OF THE HIGH-RISK HPV E7 ONCOPROTEINS

HPV types associated with genital disease are termed "high-risk" or "low-risk" viruses according to their prevalence in cancers. Two viral genes, E6 and E7, are invariably expressed in cervical carcinomas. The E7 gene product has been found to bind the retinoblastoma tumor suppressor protein and to be phosphorylated by casein kinase II. Although present in both high- and low-risk E7 proteins, these activities are diminished in the low-risk HPV-6 E7 polypeptide. To better understand the oncogenic potential of the HPV-6 E7 protein, we replaced four of its amino acids with HPV-16 E7 residues present in the analogous region of the N-terminal half of the protein. Replacement of the arginine at position 4 of the HPV-6 E7 protein with an aspartate present in HPV-16 E7 slowed the mobility of the protein when expressed in vivo. Replacement of the glycine at position 22 with an aspartate resulted in higher affinity for retinoblastoma protein binding. Replacement of valine residues at positions 30 and 37 with asparagine and aspartate, respectively, resulted in higher levels of casein kinase II phosphorylation. The substitution at position 22 was the only mutation that exhibited increased transforming activity, suggesting a correlation between the HPV E7 protein affinity for the retinoblastoma tumor suppressor protein and its ability to transform established cells. Our results show that subtle changes in sequence may result in marked differences in biological activity of HPV oncogenes.