THE FUNCTIONS OF HUMAN PAPILLOMAVIRUS TYPE-11 E1, E2, AND E2C PROTEINS IN CELL-FREE DNA-REPLICATION

We examined the functions of human papillomavirus type 11 (HPV-11) El and E2 proteins purified from Sf9 cells infected with recombinant bacu loviruses in cell-free HPV-11 origin (ori) replication. The El protein binds specifically to a wild type but not tb a mutated sequence in th e ori spanning nucleotide position 1. It also has a relatively strong affinity for nonspecific DNA, A neutralizing antiserum directed agains t the amino-terminal one-third of the El protein totally abolishes ini tiation and elongation, suggesting that it functions as an initiator a nd a helicase at the replication fork. An antiserum against the carbox yl-terminal portion of El protein abolished replication only when adde d prior to initiation, Thus this portion of El is hidden in the replic ation complexes. The HPV-11 E2 protein appears not to be essential for elongation, but it must be present in the preinitiation complex for t he El protein to recruit host DNA replication machinery to the ori. E2 antiserum added after preincubation in the absence of the cell extrac ts totally abolished replication, An identical conclusion is also reac hed for the bovine papillomavirus type 1 E2 protein. Finally, the HPV- 11 E2C protein lacking the transacting domain of the full-length E2 pr otein partially inhibits E2-dependent ori replication.