The large family of human papillomaviruses (HPVs) ()cause persistent or recurrent epitheliotropic lesions, some of which can progress to high grade dysplasias or carcinomas(1) . Productive infections normally cause exophylic or flat warts in which the viruses have two distinct modes of DNA replication. A low copy number of viral DNA is maintained in basal and parabasal cells that are capable of cell division. Only in a subset of cells undergoing terminal squamous differentiation does vegetative viral DNA amplification take place(2) . There is considerable interest in investigating the mechanisms of papillomaviral DNA replication; they may serve as models for host DNA replication as alternatives to SV40 and polyomavirus, and means might be identified to suppress or eradicate persistent infections. However, because of their stringent dependence on squamous epithelial differentiation, these viruses cannot be propagated in cells cultured by conventional means, making it difficult to investigate viral DNA replication.

Two assays have been developed to study DNA replication of HPVs and bovine papillomavirus type 1 (BPV-1) which cause fibropapillomas in cattle. One is transient replication of plasmids in mammalian cells cotransfected with expression vectors for viral genes. The other is cell-free replication in the presence of viral proteins purified from insect Sf9 cells infected with recombinant baculoviruses. These assays have demonstrated that replication requires a viral origin of replication (ori), virus-encoded E1 and E2 proteins and the host DNA replication enzymes including DNA polymerase /primase, proliferating cell nuclear antigen/DNA polymerase or , single-stranded DNA binding protein RPA, and topoisomerases I and II ((3, 4, 5, 6, 7, 8, 9) ; for a review, see (10) ).

The full-length E2 proteins of HPVs and BPV-1 are also 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 (11) ). They bind as a dimer to a consensus sequence ACCNGGT designated the E2-BS(12, 13, 14, 15, 16) . The full-length HPV-11 E2 protein is the primary HPV-11 ori binding protein (8, 17) (see below). Two alternative HPV-11 E2 proteins, E2C and E1ME2C, 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) . The simplest explanation would be the inefficiency of cotransfection of four plasmids into the same cells by using electroporation. This hypothesis remains to be tested.

Aside from the ability of the purified HPV-11 E1 protein to support cell-free replication(8) , the E1 proteins of various HPV types are only partially characterized. A bacterially expressed maltose/HPV-6b E1 fusion protein exhibits helicase activity(20) . A glutathione S-transferase fusion protein with the HPV-31b E1 protein purified from bacteria binds to a sequence that has some homology to and is located at a similar genomic position as the BPV-1 E1 binding site (E1-BS) in the BPV-1 ori(21) . Using crude cell extracts of Sf9 cells infected with recombinant baculovirus, the ability of the HPV-11 E1 to bind to an ori fragment has been demonstrated by an immunoprecipitation assay(22) . However, in another report, sequence-specific binding of HPV-11 E1 protein partially purified by immunoprecipitation from Sf9 cells was not observed, although the protein exhibited ATPase and GTPase activities(23) . A caveat of these binding results is that the functionality of the E1 protein was not corroborated by cell-free replication assays, nor was mutational analysis of the putative E1-BS performed.

In contrast, the BPV-1 E1 protein, which is highly homologous to the HPV E1 proteins, is well characterized. It is a DNA-dependent ATPase and an ATP-dependent helicase(24, 25, 26) . Unlike the HPV systems, it is the major BPV-1 ori recognition protein and binds to an 18-bp imperfect palindrome spanning nucleotide 1 (E1-BS)(3, 27, 28, 29, 30) . As does the SV40 T antigen, the E1 protein distorts and melts the ori upon binding (31) and interacts with the DNA polymerase (32, 33) . In addition, it interacts with the BPV-1 E2 protein in the presence or in the absence of the respective cognate binding sites(3, 31, 34, 35, 36, 37, 38, 39) . BPV-1 E1 protein formed two different complexes around the BPV-1 ori(40) . At a high E2/E1 ratio where replication was inhibited, a c1 complex was formed that contained both BPV-1 E1 and E2 proteins. In contrast, at a low E2/E1 ratio when replication took place, a c2 complex was formed that contained no E2 protein. It was proposed that the E2 protein helps recruit and stabilize the multiple copies of E1 protein to the ori and then has to be released before initiation of DNA replication can occur. This hypothesis has not been tested experimentally.

All papillomaviruses have multiple copies of E2-BS in the upstream regulatory region (or long control region) that contains the ori(3, 5, 6, 7, 28) . Site-directed mutagenesis demonstrated that one or more copies of the natural E2-BS are absolutely necessary for HPV-11 replication proteins to initiate ori-specific replication in both transient and cell-free replication assays(8, 17) . Conversely, the presence of one or more copies of synthetic E2-BS are sufficient to initiate replication by HPV-11 or HPV-18 replication proteins in either assay(22, 41) . ()A putative E1-BS in the HPV-11 ori greatly enhances the efficiency of cell-free replication, whereas transcriptional enhancer elements have little effect in transient or cell-free replication assays(43) . The natural oris of HPV-11 and BPV-1 also each contains an AT-rich region proximal to the E1-BS(3, 17, 28) . However, in the absence of the E1-BS, this element had little contribution during cell-free ori replication by HPV-11 proteins.

In this report, we examined the roles of the HPV-11 E1, E2 and E2C proteins in the HPV ori-specific replication. Each protein was purified from Sf9 cells infected with a recombinant baculovirus. We show that the replication-competent HPV-11 E1 protein is able to bind specifically to the ori at the previously inferred E1-BS. It also exhibits a relatively strong nonspecific affinity for DNA. We show that E1 protein is required for both initiation and elongation but that only a small proportion of the daughter molecules entered into a second round of replication. Our results also show that E2 protein appears not to be essential for elongation. We detected no replication-competent HPV-11 or BPV-1 E1DNA complexes that were not sensitive to neutralizing anti-E2 antisera. We also show that HPV-11 E2C protein can inhibit HPV-11 ori replication, but inhibition was not very effective, even in the presence of a large excess of E2C protein.

MATERIALS AND METHODS

Origin Plasmids

Viral Proteins

Cell-free Replication Assay

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Analysis of Replication Products by Digestion with DpnI Restriction Endonuclease

Electrophoretic Mobility Shift Assay (EMSA)

Immunoprecipitation

RESULTS

HPV-11 E1 Protein Is a DNA Binding Protein

Figure 1: HPV-11 E1 protein has specific and nonspecific DNA affinity. DNA binding of HPV-11 E1 protein was tested by electrophoretic mobility shift assays and by immunoprecipitation assays. A, a P-end-labeled DNA fragment containing HPV-11 sequence 7874-7933/1-20 generated by polymerase chain reaction was used as a probe (lane 1). HPV-11 E1 protein (240 ng) was mixed with probe in the absence (lane 2) or presence (lanes 3-6) of 4 mM ATP. One µl of purified anti-EE antibody was added in the reaction of lane 4. Competitor A was the unlabeled, homologous DNA fragment. Competitor B has two mutations creating an HpaI site spanning nt position 3 (oriH).B, a P-end-labeled wild type probe (lanes 7-9) or oriH probe (lanes 10-12) generated by restriction digestions was used in mobility shift assays with 240 ng of HPV-11 E1 proteins (lanes 8 and 11) or 80 ng of HPV-11 E2C protein (lanes 9 and 12). A small fraction of the fragments generated dimers and trimers through the cohesive ends. C, immunoprecipitation assays were conducted by mixing 240 ng of E1 protein (lane 15) or 80 ng of HPV-11 E2 protein (lane 14). End-labeled DNA fragments used for binding are shown in lane 13. Lane 14, immunoprecipitation with E2 antibody. Lane 15, immunoprecipitation with anti-EE antibody. Specific fragments containing HPV-11 E1-BS (7730-99-234M and 7874-99) or E2-BS (7874-99 and SN3) are indicated on the right, and sizes of the DNA fragments (in base pairs) are indicated on the left.

The E1/E1-BS interaction is however relatively weak, as the complexes were eliminated by poly(dI-dC) at more than 150-fold mass excess and were reduced at NaCl concentration higher than 25 mM. No complexes were detected when glutaraldehyde cross-linking was omitted (data not shown). The E1 protein also had relatively strong nonspecific DNA binding ability. In the absence of poly(dI-dC), only large complexes that did not enter into the gel were formed with DNA containing either wild type or mutated E1BS (data not shown). In the presence of a 100-fold mass excess of poly(dI-dC) where specific binding to the E1-BS was observed, much of these large complexes remained (Fig. 1A).

This relatively small difference in the affinity of E1 protein for E1-BS and for nonspecific DNA sequence was also manifested in the immunoprecipitation assays (Fig. 1C). Using the anti-EE Mab to precipitate the E1DNA complex, there was an enrichment of the two E1-BS-containing fragments, 7874-99 (which contains the E1-BS and three copies of natural E2-BS) and 7730-99-234M (which contains the E1-BS but mutated E2-BS), over the nonspecific fragments from pUC-19 (lane 15), but only over a very narrow range of binding conditions (data not shown). Polyclonal anti-E1 antisera disrupted the complex and led to nonspecific precipitation of all fragments (data not shown). In parallel experiments under the same binding conditions, anti-E2 antiserum precipitated only fragment 7874-99 and SN3 that contain three copies of synthetic E2-BS in association with HPV-11 E2 protein but not the other fragments (lane 14). The fragment 7730-99-234M, in which all three E2-BS were mutated, has also been shown previously not to bind E2C protein by DNase I footprinting assay(17) .

HPV-11 E1 Protein Is Necessary for both Replication Initiation and Elongation but HPV-11 E2 Protein Appears Not to Be Essential for Elongation

Figure 3: HPV-11 E1 protein is relatively insensitive to neutralizing E1C antiserum during elongation. A, time course of antisera addition. The experiments were similar to those described in the left panel of Fig. 2except that [-P]dCTP was added at 30 min after the initiation of incubation. Rabbit polyclonal anti-E1N and anti-E2 antisera, as well as antiserum raised against Trp E fused with the carboxyl-terminal 402 amino acids of HPV-11 E1 (E1C) were added at different times postinitiation. B, autoradiogram of cell-free replication of ori plasmid pUC7874-99 conducted with no viral proteins (lane 1), or with 30 ng of HPV-11 E1 and 8 ng of HPV-11 E2. Lane 2, no antiserum added. Lanes 3-20, 1 µl of anti-E1N or anti-E1C or 2 µl of anti-E2 antiserum as indicated was added at times shown above the lanes. SV40 cell-free replication with 40 ng of pSVori, 40 ng of T antigen, and 10 µl of 293 cell extracts (lane 21) was not affected significant by anti-E1N (lane 22) or anti-E2 (lane 23) antiserum. Migration of replication intermediates and Forms I and II DNA is marked on both sides. Quantitative analyses of total incorporation (C) and Form I DNA (D) during HPV-11 ori replication by PhosphorImager are shown.

Figure 2: HPV-11 E1 protein is required for both initiation and elongation, whereas the HPV-11 E2 protein appears to be required only for initiation. A, time course of antisera or stop solution addition. Each reaction contained 40 ng of ori plasmid pUC7874-99, 30 ng of HPV-11 E1 protein, 8 ng of HPV-11 E2 protein, and 10 µl (10 mg/ml) of 293 cell extract in reaction buffer at time 0. Fifteen min into the reaction, 2.5 µCi of [-P]dCTP were added. Anti-E1N and anti-E2 rabbit polyclonal antisera used were raised against the amino-terminal portion of the HPV-11 E1 protein (5) or the HPV-11 E2 protein(19) . Antisera (left panel) or stop solution (right panel) was added at different times as indicated by the arrow, and the reactions continued until termination at 105 min by the addition of stop solution. B, total incorporation into replication products (minus repair synthesis) were quantified by PhosphorImager for the three experiments. C, incorporation into RI or Form I molecules in the presence of E1N or E2 antiserum after subtracting the counts in the control experiment generated by the addition of stop solution at each time point.

The experimental scheme is illustrated in Fig. 2A. E1- or E2-specific antiserum was added postinitiation at various times after the addition of [-P]dCTP. Incubation was allowed to continue to 105 min and was then terminated by the addition of the stop solution of SDS and proteinase K. The extent of replication that took place up to the moment of antisera addition was determined in parallel experiments by the addition of stop solution (Fig. 2A, right panel). We reasoned that if the viral proteins are required for both initiation and elongation, the addition of neutralizing antisera should, ideally, block further replication as effectively as the stop solution. The quantitative analyses of total incorporation (Fig. 2B) of all three sets of reactions (after subtraction of repair synthesis) were determined by PhosphorImager. The amounts of Form I and replication intermediate (RI) accumulated after the addition of E1N antiserum or E2 antiserum in excess over the control experiments are shown in Fig. 2C. The E1N antiserum virtually abolished all subsequent [-P]dCTP incorporation into both RI and Form I molecules, indicating that E1 is required for both initiation and elongation and must remain in active form in association with the replication intermediates until the completion of replication. In contrast, the addition of E2 antiserum did not prevent the incorporation of [-P]dCTP into RI or Form I DNA. We interpret these results to mean that elongation was not inhibited, at least not extensively, by E2 antiserum, and consequently the E2 protein appears not to be essential for elongation. The accumulation of RI increased for 30 min and then steadily decreased, whereas Form I DNA plateaued at 60-75 min. This delayed accumulation of Form I relative to RI is consistent with a product-precursor relationship. Furthermore, the decline in RI is in dramatic contrast to their continued accumulation in the absence of antisera(8) . These results suggest that RI that matured into Form I DNA was not replenished by newly initiated RI in the presence of E2 antiserum. We concluded that E2 antiserum inhibits initiation but not elongation (see also Fig. 3).

Nonspecific inhibition of replication by either antisera was ruled out on the basis of several observations. Preimmune serum or serum raised against Trp E protein that is present in the immunizing antigens (TrpE-E1N or TrpE-E2), had little or no effect on cell-free replication (data not shown), nor did a polyclonal antiserum against a Trp E-HPV-11 E5a fusion protein(8) . Neither antiserum significantly affected cell-free SV40 replication initiated by the SV40 T antigen (Fig. 3B, lanes 21-23). Moreover, the E1N and E2 antisera each detected, in Western blots, a single protein band in COS cells transfected with E1 or E2 expression plasmids(5) .

Postinitiation Replication Complexes Are Relatively Insensitive to HPV-11 E1C Antiserum

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HPV-11 E2 Protein Is Required during Assembly of Preinitiation Complexes

Figure 4: HPV-11 E2 protein is required for assembly of preinitiation complexes. A, time course of addition of cell-free reaction components. 40 ng of pUC7874-99, 30 ng of HPV-11 E1, 8 ng of HPV-11 E2, and all eight nucleoside triphosphates were mixed in reaction buffer and preincubated at 37 °C for 30, 20, 10, or 0 min(-30, -20, -10, 0). Two µl of HPV-11 E2 antiserum or buffer were added together with 10 µl of 293 cell extracts at time 0. [-P]dCTP was added at 45 min, and reactions were terminated at 90 min by stop solution. B, autoradiogram of the experiments described in panel A. The presence (+) or absence(-) of viral proteins or E2 antiserum is indicated at the top of each lane.

BPV-1 E2 Protein Is Required during Assembly of Preinitiation Complexes

Figure 5: BPV-11 E2 protein is required for assembly of preinitiation complexes. A, time course of addition of cell-free reaction components. The experiment had the same design as that in Fig. 4except [-P]dCTP was added at 30 min and the reaction was terminated at 60 min. 40 ng of BPV-1 ori plasmid pUC-Alu, 90 ng of BPV-1 E1, 30 ng of BPV-1 E2 proteins, and eight nucleoside triphosphates in reaction buffer were preincubated for 30, 20, 10, or 0 min (-30, -20, -10, and 0 min) as indicated. One-half µl of BPV-1 E2 antiserum or buffer was added at time 0. B, autoradiogram of the experiments described in panel A. The presence (+) or absence(-) of viral proteins or E2 antiserum is indicated at the top of each lane.

Figure 6: HPV-11 E2C protein is a replication repressor. A, autoradiogram of cell-free replication in the presence of 30 ng of E1, 8 ng of E2 proteins, and different amounts (in ng) of HPV-11 E2C protein using as template the HPV-11 ori plasmid pUC7874-20 (lanes 1-5), which contains one copy of E1-BS, or pUC7874-99 (lanes 6-10), which contains three copies of E2-BS. Lanes 1 and 6 were reactions in the absence of viral protein. B, SV40 cell-free replication with 40 ng of T antigen, 40 ng of pSVori plasmid, and 10 µl of 293 cell extracts was used to rule out the nonspecific effect of HPV-11 E2C protein fraction. C, total incorporation (minus repair synthesis) in all three ori plasmids was quantified by PhosphorImager. Reaction without E2C protein was taken to be 100%

E2C Is a Negative Factor in HPV-11 Cell-free Replication

Cell-free Replication Products Are Mostly Semimethylated Resulting from a Single Round of Semiconservative Replication

The results are shown in Fig. 7A. All of the digestions were complete (compare lane 3 to lanes 2 and 4-7). PhosphorImager recording revealed P-labeled molecules before and after digestions (compare lane 9 to lanes 8 and 10-13). Undigested replication products migrated as Form II, various topoisomers of Form I, and slow migrating replication intermediates (lane 9) as shown previously (8) and throughout this study (Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6). Digestion with EcoRI alone, which cuts once in the polylinker just outside the ori fragment at nt 396 (see Fig. 7B), generated P-labeled, unit-length linear DNA (Form III) (lane 10) derived from the Form I and Form II DNA. The partially replicated intermediates were converted to slow migrating molecules attributable to their longer than unit length and forked ends after digestion of form molecules. When the replication products were digested with both EcoRI and 0.5 or 1 unit of DpnI, much of the Form III-labeled DNA remained (lanes 11 and 8). The replication intermediates were converted to fragments shorter than unit length, most of which were, however, longer than the largest unlabeled fragment a of 1,100 bp generated from a complete digestion of the carrier DNA (compare lanes 8 and 11 with lanes 2 and 5-7; see also panel B). A small amount of slow migrating material persisted, probably for reasons stated above. Thus, these results confirm that both the P-labeled Form I and the slow migrating molecules generated in the replication reaction are products of replicative DNA synthesis rather than repair synthesis, which would have yielded small fragments similar to those shown in lanes 5-7 upon DpnI digestion. Increasing amounts of DpnI to 2 or 4 units decreased Form I DNA and also correspondingly reduced the sizes of the majority of the other P-labeled materials (lanes 12 and 13); the pattern of digestion remained the same at as high as 10 units of DpnI (data not shown). These results suggest that most of the replicated DNA has gone through only one complete or partial round of semiconservative replication and that few have reinitiated from the daughter molecules producing unmethylated DNA. Of note is that the most heavily labeled 1,100-bp fragment a in the complete digestion by excess DpnI (lane 13) contains the HPV-11 ori insert. The next most heavily labeled 585-bp fragment b was close the ori fragment (with the small DpnI fragment between fragments a and b cut into two by EcoRI and lost from the gel during migration or drying) (Fig. 7B). These results are consistent with the interpretation that replication initiated within the 1,100-bp fragment containing the HPV-11 ori.

Figure 7: HPV-11 ori-specific replication product is resistant to DpnI digestion but sensitive to excess enzyme and approximate localization of ori. A, cell-free replication using 40 ng of HPV-11 ori plasmid pUC7874-99/reaction was carried out in the presence of [-P]dCTP. The purified DNA products from six such reactions were mixed with 6 µg of unlabeled template carrier, equally divided into six aliquots, and then treated as described below. Lanes 1-7, ethidium bromide-stained gel prior to recording by PhosphorImager (lanes 8-13). Lane 1, 1-kb ladder size marker (Life Technologies, Inc.). Lanes 3 and 9, undigested DNA. Lanes 4 and 10, digestions with 4 units of EcoRI alone. All other lanes were digested with 4 units of EcoRI plus different amounts of DpnI as follows: lanes 2 and 8, 1 unit; lanes 5 and 11, 0.5 unit; lanes 6 and 12, 2 units; lanes 7 and 13, 4 units. Fragments a and b are the most heavily labeled, indicative of their proximity to ori. B, the location of HPV-11 ori insertion relative to DpnI cut sites that generated fragments a and b. EcoRI cuts at nt 396, reducing the small fragment between fragments a and b to 22 bp and 119 bp. These small fragments were lost from the gel during migration or drying.

DISCUSSION

Using HPV-11 E1 and E2 proteins purified from Sf9 cells, we have examined the roles of both proteins in HPV-11 ori replication. We show that E1 protein binds to a sequence in the HPV-11 ori spanning nucleotide 1 (designated E1-BS) only in the presence of ATP by EMSA and the immunoprecipitation assays (Fig. 1). The HPV-11 E1-BS is analogous in location and in nucleotide sequence to the E1-BS in BPV-1 ori. All the papillomaviruses appear to have this putative E1-BS at the same genomic location(10) . This ability of HPV-11 E1 protein to bind to the E1-BS explains the 6-fold stimulation of HPV-11 E2-dependent ori activity by the presence of E1-BS observed in cell-free replication. It may also contribute to the E2-independent ori replication promoted by high concentrations of E1 protein in the cell-free system(8) . The HPV-1 and BPV-1 E1 proteins also exhibited E2-independent replication in transient replication or cell-free replication, presumably through the same mechanism(9, 24, 33, 39, 49, 50) .

Our experiments with neutralizing antiserum to the amino-terminal portion of the E1 protein demonstrate that E1 protein is required not only for initiation but also for elongation, as the addition of the antiserum completely prevented initiation and caused an immediate cessation of elongation ( Fig. 2and Fig. 3). The BPV-1 and HPV-6 E1 fusion proteins are helicase(20, 24, 25) . Together with the results just described, the E1 protein of HPVs and BPV-1 most likely remains at the replication forks, unwinding the parental DNA strands in an ATP-dependent manner as elongation proceeds. A similar conclusion has been reached for the SV40 T-ag (for a review, see (51) ). This interpretation is consistent with the relatively strong nonspecific DNA affinity of E1 protein (Fig. 2), as may be expected for a helicase required throughout the replication reaction. In turn, this nonspecific DNA binding could account for the ori-independent cell-free replication by high concentrations of HPV-11 or BPV-1 E1 protein(8, 24, 33, 39, 49, 50) . Identical experiments with antisera against BPV-1 E1 protein in BPV-1 ori replication, however, did not lead to a complete cessation of replication, perhaps because the particular antiserum did not recognize the vulnerable region of the E1 protein (data not shown). Interestingly, using DpnI sensitivity as a probe, we demonstrated that most of the replication products resulted from one round of semiconservative replication, which initiated from sequences containing the HPV-11 ori, generating semimethylated daughter molecules (Fig. 7). Although the majority of the replication products were resistant to DpnI digestion, they were sensitive to excess enzyme. Thus the E1 protein dissociates from the finished products and then reinitiates from different template molecules. Alternatively, the majority of viral or host proteins became inactivated during incubation, and thus little reinitiation occurred. These possibilities remain to be examined.

The polyclonal antiserum against the carboxyl of the HPV-11 E1 protein did not completely inhibit replication when it was added postinitiation. However, complete inhibition was observed when it was added prior to initiation (Fig. 3, B and C). We infer that the amino-terminal portion of the E1 protein remains relatively exposed and accessible to antiserum inactivation throughout initiation and elongation, whereas the carboxyl-terminal portion including the predicted ATP binding and ATPase domains becomes partially hidden in the elongation complex due to oligomerization of the E1 protein or to interaction with host replication enzymes. SV40 T antigen functions as a double hexamer at the replication fork(52) . The c2 complex of the BPV-1 E1 protein is also proposed to be a hexamer (40) . Interestingly, the monoclonal antibody against the EE-epitope on the E1 protein had very little effect when added during preincubation or postinitiation (data not shown), indicating that the very N terminus of the E1 protein was not in functional contact with viral and host replication proteins, nor with the DNA template.

Unlike the anti-E1N antiserum, the addition of HPV-11 E2 antiserum resulted in total inhibition of replication only when the antiserum was added prior to but not after initiation ( Fig. 2and Fig. 3). There are two possible explanations. We favor the interpretation that E2 protein is needed only for initiation and that it is not present in the elongation complexes. This interpretation is consistent with the continued accumulation of Form I daughter molecules after antiserum addition (Fig. 2C) and the ability of high concentrations of E1 protein alone to initiate replication from ori or ori templates(8) . An alternative possibility that cannot be formally ruled out is that E2 protein is present in the elongation complexes but becomes less accessible to the antiserum than when it is in the preinitiation complexes.

We also demonstrated that the presence of E2 protein of HPV-11 or BPV-1 in the preinitiation complex is absolutely necessary, as the addition of HPV-11 or BPV-1 E2 antiserum to the mixture of DNA and viral proteins after a 30-min preincubation in the absence of cell extracts totally abolished HPV-11 or BPV-1 ori replication ( Fig. 4and Fig. 5). We suspect that the E2 protein not only stabilizes E1 binding to the ori(3, 28, 37, 38, 39) , it also interacts with host DNA replication machinery during the assembly of the preinitiation complexes. The BPV-1 E2 protein binds weakly to the single-stranded DNA binding protein RPA(53) . Considering the functional similarities of the E2 proteins among animal and human papillomavirus, it is very likely that HPV-11 E2 also has a similar activity. Alternatively, E2 may aid indirectly the recruitment of host replication proteins to the ori by the E1 protein. Binding of E2 proteins introduces a sharp bend into the DNA(54, 55) . This change of DNA conformation may increase E1/E1-BS affinity, stabilize E1/host protein interactions, or facilitate ori unwinding.

The role of E2 in transient replication cannot be fulfilled by the E2C protein devoid of the transacting domain; rather it inhibited replication in transient assays(17) . We now have identical results using the cell-free replication assay (Fig. 6). Thus, the N-terminal domain of the intact E2 protein is required for initiation of replication, perhaps through interaction with the E1 protein or the host replication machinery. Interactions between BPV-1 E1 and E2 proteins in the presence or in the absence of an ori have been reported(3, 31, 34, 35, 36, 37, 38, 39) . Presently, we have not been able to demonstrate any interaction between HPV-11 E1 and E2 proteins under the conditions described in Fig. 1where specific interactions between DNA and each protein were detected, probably due to a very weak affinity between the two proteins and between the E1 and E1-BS. The relatively strong E1 affinity for nonspecific DNA also presented additional difficulties. However, the transient replication assay with matched or mixed pairs of E1 and E2 proteins from different papillomaviruses suggest that such an interaction must exist to account for the differential ability to replicate an ori consisting of synthetic E2-BS alone or a natural ori with both E2-BS and E1-BS. In principle, the E2C might inhibit replication by competing with the full-length E2 for the E2-BS that serves as ori. E1-E2 protein interactions may increase the affinity of E2 for the E2-BS, as concluded for BPV-1 proteins(3, 37, 39, 42) , whereas E2C may not be able to do so. This enhanced E2 affinity for E2-BS relative to E2C could account for a high ratio of E2C to E2 to effect inhibition. Alternatively, heterodimers of E2 and E2C may support replication, as observed with BPV-1 E2 proteins. ()We have no information about whether such heterodimers could form under our cell-free replication conditions.

In summary, we have used cell-free replication to investigate the functions of papillomavirus E1 and E2 proteins. We show that the E1 protein of HPV-11 is required for initiation and elongation, that HPV-11 E2 is not essential for elongation, and that the HPV-11 E2 protein and the BPV-1 E2 protein are each required for the assembly of preinitiation complex for HPV-11 ori or BPV-1 ori replication. The HPV-11 E2C protein is capable of inhibiting ori replication, albeit very inefficiently.

FOOTNOTES

*

This work was supported by U.S. Public Health Service Grant CA36200. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, 1918 University Blvd., BHSB Room 510, Birmingham, AL 35294-0005. Tel.: 205-975-8300; Fax: 205-975-6075.

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The abbreviations used are: HPV, human papillomavirus; BPV, bovine papillomavirus; bp, base pair(s); nt, nucleotide(s); EMSA, electrophoretic mobility shift assay; RI, replication intermediate.

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J.-S. Liu, S.-R. Kuo, T. R. Broker, and L. T. Chow, submitted for publication.

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T. F. Ho, J.-S. Liu, L. T. Chow, and T. R. Broker, unpublished results.

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J.-S. Liu, S.-R. Kuo, T. R. Broker, and L. T. Chow, unpublished results.

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D. A. Lim and M. R. Botchan, personal communication.

ACKNOWLEDGEMENTS

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