Human papillomavirus type 31 oncoproteins E6 and E7 are required for the maintenance of episomes during the viral life cycle in normal human keratinocytes

The E6 and E7 oncoproteins of the high-risk human papillomavirus (HPV) type s are able to immortalize human keratinocytes in vitro and likely contribut e to the development of anogenital malignancies in vivo. The role of these oncoproteins in the productive viral life cycle, however, is not known. To begin to examine these possible roles, mutations in E6 were introduced in t he context of the complete HPV 31 genome. Although transfected wild-type HP V 31 genomes, as well as genomes containing an E6 translation termination l inker, an E6 frameshift mutation, and a point mutation in the p53 interacti ng domain were able to replicate in transient assays, only the wild-type ge nome was stably maintained as an episome, Interestingly, mutant genomes in either the E6 splice-donor site or splice-acceptor site were reduced in rep lication ability in transient assays; however, cotransfection of E1 and E2 expression vectors restored this function. In a similar fashion, genomes co ntaining mutant HPV 31 E7 genes, including a translation termination mutant , two Rb-binding site mutants, a casein kinase II phosphorylation site muta nt, and a transformation deficient mutant, were constructed, Although trans ient replication was similar to wild type in all of the E7 mutants, only th e casein kinase II mutant had the ability to maintain high copies of episom al genomes, These findings suggest a role for E6 and E7 in the viral life c ycle beyond their ability to extend the life span of infected cells.