TRANSACTIVATION BY THE E2 PROTEIN OF ONCOGENIC HUMAN-PAPILLOMAVIRUS TYPE-31 IS NOT ESSENTIAL FOR EARLY AND LATE VIRAL FUNCTIONS

The activation of transcription and of DNA replication are, in some ca ses, mediated by the same proteins. A prime example is the E2 protein of human papillomaviruses (HPVs), which binds ACCN(6)GGT sequences and activates heterologous promoters from multimerized binding sites. The E2 protein also has functions in replication, where it complexes with the virally encoded origin recognition protein, El. Much of the infor mation on these activities is based on transient-transfection assays a s well as biochemical analyses; however, their importance in the produ ctive life cycle of oncogenic HPVs remains unclear. To determine the c ontributions of these E2 functions to the HPV life cycle, a genetic an alysis was performed by using an organotypic tissue culture model. HPV type 31 (HPV31) genomes that contained mutations in the N terminus of E2 (amino acid 73) were constructed; these mutants retained replicati on activities but were transactivation defective. Following transfecti on of normal human keratinocytes, these mutant genomes were establishe d as stable episomes and expressed early viral transcripts at levels s imilar to those of wild-type HPV31. Upon differentiation in organotypi c raft cultures, the induction of late gene expression and amplificati on of viral DNA were detected in cell lines harboring mutant genomes. Interestingly, only a modest reduction in late gene expression was obs erved in the mutant lines. We conclude that the transactivation functi on of E2 is not essential for the viral life cycle of oncogenic HPVs, although it may act to moderately augment late expression. Our studies suggest that the primary positive role of E2 in the viral life cycle is as a replication factor.