Abstract
We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cell-free HPV-11 origin (ori) replication. The E1 protein binds specifically to a wild type but not to a mutated sequence in the ori spanning nucleotide position 1. It also has a relatively strong affinity for nonspecific DNA. A neutralizing antiserum directed against the amino-terminal one-third of the E1 protein totally abolishes initiation and elongation, suggesting that it functions as an initiator and a helicase at the replication fork. An antiserum against the carboxyl-terminal portion of E1 protein abolished replication only when added prior to initiation. Thus this portion of E1 is hidden in the replication complexes. The HPV-11 E2 protein appears not to be essential for elongation, but it must be present in the preinitiation complex for the E1 protein to recruit host DNA replication machinery to the ori. E2 antiserum added after preincubation in the absence of the cell extracts totally abolished replication. An identical conclusion is also reached for the bovine papillomavirus type 1 E2 protein. Finally, the HPV-11 E2C protein lacking the transacting domain of the full-length E2 protein partially inhibits E2-dependent ori replication.
Highlights
We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cellfree human papillomaviruses (HPVs)-11 origin replication
HPV-11 E1 Protein Is a DNA Binding Protein—Since a mutation in a putative HPV-11 E1 binding site (E1-BS) in the HPV-11 ori inferred from a partial sequence homology to the bovine papillomavirus type 1 (BPV-1) E1-BS greatly reduced the efficiency of cell-free replication,2 we examined the possibility that the HPV-11 E1 protein binds to this sequence
Using HPV-11 E1 and E2 proteins purified from Sf9 cells, we have examined the roles of both proteins in HPV-11 ori replication
Summary
We examined the functions of human papillomavirus type 11 (HPV-11) E1 and E2 proteins purified from Sf9 cells infected with recombinant baculoviruses in cellfree HPV-11 origin (ori) replication. The full-length E2 proteins of HPVs and BPV-1 are transcription regulatory proteins, each consisting of three domains, the amino-terminal transactivating domain, a hinge region, and the carboxyl-terminal DNA binding and protein dimerization domain (for a review, see Ref. 11). They bind as a dimer to a consensus sequence ACCN6GGT designated the E2-BS [12,13,14,15,16]. Two alternative HPV-11 E2 proteins, E2C and E1M∧E2C, encoded by separate mRNAs contain the hinge and the DNA binding and dimerization domains but have different amino termini Both are strong transcription repressors but are weak repressors in transient replication [17,18,19]. Unlike the HPV systems, it is the major BPV-1 ori recog-
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