Centre for Biomolecular Sciences, School of Biology, University of St Andrews, Biomolecular Sciences Building, North Haugh, St Andrews, Fife KY16 9ST, UK1
Author for correspondence: Ronald Hay. Fax +44 1334 462595. e-mail rth{at}st-andrews.ac.uk
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Abstract |
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Introduction |
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Previous attempts to make mutations in pTP have been confined to linker insertion mutagenesis and deletion of residues from the N terminus of pTP (Fredman et al., 1991 ; Freimuth & Ginsberg, 1986
; Pettit et al., 1989
; Roovers et al., 1991
, 1993
; Schaack et al., 1999
). Introduction of such mutations into the virus genome and transfections into a pTP complementing cell line allowed the growth of virus containing these mutations. A number of the mutants made proved to be silent showing that it is possible to mutate pTP by the addition of insertions and maintain activity. However, many viruses containing these mutations were not infectious in non-complementing cell lines. The phenotypes of the remaining mutants indicated the important role of specific pTP regions in viral infectivity.
Although there is as yet no structural information available for pTP the domain organization of the protein and the roles of different regions of the protein have been elucidated (Webster et al., 1997a ). The sites of interaction of a panel of monoclonal antibodies raised against pTP were identified. These largely coincide with the regions most susceptible to proteolysis, thus defining domain junctions or relatively unstructured regions. The Adpol-binding region was mapped to the C-terminal 60 kDa of the protein, whilst the N-terminal portion was involved in DNA binding. However, other portions of the molecule contribute to the strength of the interactions. It was shown that conformational antibodies against pTP disrupted DNA binding and could displace Adpol, highlighting the importance of protein flexibility in the interactions of pTP.
Here we use site-directed mutagenesis of pTP to define the roles of individual amino acids, conserved between the various strains of adenovirus and predicted to be surface exposed, in DNA replication.
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Methods |
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The N-terminal deletions of aa 1175, giving iTP, and of aa 1349, giving TP, were made by PCR using forward primers 39 and 40, containing a BamHI site, and reverse primer 6, containing a KpnI site. The PCR product was digested with BamHI and KpnI and ligated with cut pGEM-3Z vector. For all the mutants, the sequence of the inserted region was confirmed by automatic DNA sequencing (Alex Houston, St Andrews University DNA sequencing service).
The mutated genes were cut out of pGEM-3Z, using the restriction enzymes BamHI and EcoRI, and subcloned into the pFASTBAC vector cut with the same enzymes. Plasmid from positive clones was transformed into DH10BAC competent E. coli. Recombinant bacmid DNA was isolated from the positive clones and used to transfect Spodoptera frugiperda 9 (Sf9) cells and thus generate recombinant baculovirus, as described in the Bac-to-Bac baculovirus expression systems instruction manual.
Expression of pTP and mutant proteins in insect cells.
Sf9 cells (1x107) were infected with the baculovirus and harvested after 96 h. Infected cells were collected by centrifugation, washed twice with PBS (10 ml) and stored at -70 °C until required.
Cells were resuspended in 25 mM HEPESNaOH pH 8·0, 5 mM KCl, 0·5 mM MgCl2 containing protease inhibitors (Boehringer Mannheim) (1 ml) and incubated on ice for 5 min. The cells were lysed using a Dounce homogenizer and the nuclei collected by centrifugation. The cytoplasmic extract was removed and the nuclei resuspended in 25 mM HEPESNaOH pH 8·0, 1 M NaCl, 1 mM DTT containing protease inhibitors (1 ml) and incubated on ice for 15 min. The extract was clarified by high-speed centrifugation and aliquots of both nuclear and cytoplasmic extracts were snap frozen and stored at -70 °C until required. The mutants were found to be at least partially nuclear, with the exception of the aa 1349 N-terminal deletion (TP) which was totally cytoplasmic. As the nuclear extracts proved to be less sensitive to proteolysis and the protein was more soluble in high NaCl concentration conditions, nuclear extracts were used in preference to cytoplasmic extracts. However, in order to obtain stable TP and sufficient quantities of mutant 437438EE/AA and the aa 560671 C-terminal deletion, a whole cell extract was made by swelling the cells in 25 mM HEPESNaOH pH 8·0, 1 M NaCl, 1 mM DTT containing protease inhibitors directly. Extracts were clarified each time before use. All experiments were performed in duplicate.
Immunoblotting.
Samples, to which disruption buffer containing SDS and 2-mercaptoethanol was added, were denatured by heating at 100 °C for 2 min and fractionated by SDSPAGE. The proteins were electrophoretically transferred (mini Trans-Blot cell, Bio-Rad) onto PVDF membrane. Membranes were incubated in blocking buffer (PBS containing 5% non-fat milk), which was used in all subsequent incubations, and then with primary antibody at the appropriate dilution. Anti-pTP monoclonal antibodies 5E3, 44E1, 7H1 and 3D11 (Webster et al., 1997a ) were used to detect pTP/mutant pTP and an antibody raised against a peptide corresponding to the C terminus of Adpol was used to detect Adpol (Webster et al., 1994
). The membrane was washed with PBS containing 0·1% Tween 20, before antibodyantigen complexes were detected by incubation with HRP-conjugated anti-mouse or anti-rabbit antibodies, as appropriate, and, after further washing, an enhanced chemiluminescence (ECL) system.
Initiation assay.
Purified DBP (500 ng), extract prepared as described in Parker et al. (1998) from baculovirus-infected Sf9 cells containing 50 ng Adpol, extract containing 50 ng pTP/mutant pTP (dialysed against 25 mM potassium acetate, pH 8·0, 0·1 M NaCl, 1 mM DTT) and 50 ng TP-DNA template were added together with the appropriate volume of 25 mM BicineNaOH pH 8·0, 2 mM DTT, 1 mM MnCl2, 0·15 mM ATP, 0·2 mg/ml BSA to give a final volume of 10 µl. To this was added [
-32P]dCTP (2·5 µCi, 3000 Ci/mmol) and the samples were incubated at 30 °C for 1 h. Micrococcal nuclease (1 U) and CaCl2 to 10 mM were added and the samples incubated for a further 30 min at 37 °C. The reaction was stopped by the addition of disruption buffer (SDS to 2% and 2-mercaptoethanol to 0·72 M) to the reactions and the samples were denatured at 100 °C for 2 min before analysis of the reaction products by fractionation in an SDSpolyacrylamide/DATD gel; radioactivity in the dried gel was detected by PhosphorImaging.
DNA binding assay.
Extract containing 100 ng pTP/mutant pTP was added to ssDNASepharose (20 µl, 50% slurry) in 1 ml buffer containing 1% BSA (Sigma), 0·1 M (or 0·2 M) NaCl, 25 mM HEPESNaOH pH 8·0, 1 mM EDTA, 1 mM DTT, 0·05% NP40. After incubation at 4 °C with end-over-end mixing, the beads were washed once with the above buffer at the appropriate NaCl concentration, followed by three washes with the above buffer, excluding BSA, at the appropriate NaCl concentration. The samples were then transferred to fresh microcentrifuge tubes, the buffer removed and disruption buffer added. The samples were denatured by heating at 100 °C for 2 min and fractionated by SDSPAGE followed by immunoblotting, using anti-pTP monoclonal antibodies 44E1 and 3D11 as primary antibodies.
Adenovirus DNA polymerase binding assay.
Protein Gagarose beads (25 µl, 50% slurry), to which saturating levels of the monoclonal antibody 16H1 (or 3D11 in the case of the aa 183207 deletion and the aa 1349 deletion, TP) had been bound, were blocked with 1% BSA, 25 mM HEPESNaOH pH 8·0, 0·4 M NaCl, 1 mM EDTA, 1 mM DTT, 0·05% NP40 by overnight incubation at 4 °C. Extract containing 600 ng pTP/mutant pTP was added and the samples incubated for a further 2 h at 4 °C. The beads were washed three times with the above buffer and transferred to fresh siliconized microcentrifuge tubes. Extract containing 500 ng Adpol was added and the beads incubated for 2 h. They were then washed once with the above buffer and three times with the above buffer excluding BSA. The beads were transferred to fresh microcentrifuge tubes and disruption buffer was added. After heating to 100 °C proteins were fractionated by SDSPAGE, transferred to PVDF membrane and immunoblotted as described above. To assess Adpol binding, the membrane was probed with an anti-Adpol antibody as primary antibody and an anti-rabbit HRP conjugate as secondary antibody. To confirm that equal quantities of pTP/mutant pTP had bound to the beads, the membrane was stripped and reprobed with anti-pTP monoclonal antibodies and anti-mouse HRP conjugate.
Partial proteolysis.
Extract containing 130 ng pTP/mutant pTP was dialysed against 25 mM potassium acetate, pH 8·0, 0·1 M NaCl, 1 mM DTT. Each sample was normalized for total protein content by the addition of uninfected extract or the above buffer to give final volumes of 35 µl. Modified trypsin (Promega) (10 ng) was added to each sample and the samples were incubated at room temperature for 20 min. The reaction was stopped by the addition of PMSF (2·5 µl, 50 mM). Disruption buffer was added and the samples denatured by boiling and fractionated by SDSPAGE. The protein was transferred to PVDF and immunoblotted as described above, using monoclonal antibodies 3D11 and 44E1 as primary antibodies.
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Results |
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The mutations were constructed by PCR methodologies and transferred into bacmids which were used to generate recombinant baculovirus. Sf9 cells infected with recombinant baculovirus were harvested 96 h post-infection and nuclear and cytoplasmic extracts prepared. All mutants were predominantly nuclear, except the aa 1349 deletion (TP) which was totally cytoplasmic. The nuclear localization sequence (NLS) of pTP has previously been mapped to amino acids 380391 (Zhao & Padmanabhan, 1988 ), although the minimum sequence required for nuclear localization was not determined. The deletion giving TP, therefore, includes the NLS but is not transported into the nucleus in insect cells. The mutants 384385RR/AA, 386387RR/AA and 388389RR/AA have double point mutations of consecutive arginines within the predicted NLS but do not have their nuclear localization disrupted to any great degree.
As pTP was not expressed at the same level for each mutant, dilutions of cell extract from each recombinant baculovirus infection were analysed by immunoblotting using the anti-pTP monoclonal antibodies 44E1 and 3D11 (Fig. 2), and volumes of extract adjusted so that equivalent amounts of pTP mutants were added to each assay. The 484485EH/AA mutant was not expressed efficiently and therefore was not studied further.
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Ability of pTP mutants to initiate DNA replication
The ability of mutant forms of pTP to participate in viral DNA replication was determined in an in vitro initiation assay. Extract containing pTP/mutant pTP was added to an assay containing the TPviral DNA template containing the origin of replication, DBP, Adpol, Mg, ATP and dCTP. Initiation involves the Adpol-catalysed addition of [32P]dCMP to pTP. The production of [32P]pTPdCMP was assessed by separating the labelled protein from free nucleotide by SDSPAGE and analysis by autoradiography (Fig. 3). All mutants were defective for initiation compared to wild-type pTP. The mutants 356357RE/AA, 360R/A, 386387RR/AA, 520D/A and 589Q/A were reasonably efficient at initiation. Mutants 137Y/A, 139R/A, aa 183207 del, 363T/A, 388389RR/AA, 529R/A and 598599DS/AA initiated very weakly, while mutants 384385RR/AA, 437438EE/AA, 492Y/A, the C-terminal deletion, iTP (aa 1175 del) and TP (aa 1349 del) appeared not to support initiation. These results are summarized in Table 2
. Although NFIII was not added specifically to the reaction, it was expected to be present in the added extract. However, it is important to note that at the high pTP/pol concentration used the stimulatory effect of NFIII would not be observed (Coenjaerts et al., 1994
). Hence, the ability of the mutants to bind NFIII does not seem to play a major role in determining the efficiency by which they initiate adenovirus DNA replication.
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Interaction of pTP mutants with ssDNA
The ability of the mutants to bind to ssDNASepharose was assessed in the presence of either 0·1 or 0·2 M NaCl. Extracts were incubated with ssDNASepharose at the two NaCl concentrations and bound pTP was detected by immunoblotting (Fig. 4). All mutants bound DNA in 0·1 M NaCl, but carrying out the binding assay in 0·2 M NaCl allowed discrimination between the various mutants and identified some that were no longer able to bind to DNA. Those that did not bind at 0·2 M were 384385RR/AA, 386387RR/AA, 388389RR/AA and iTP (aa 1175 del). For all the mutants except 356357RE/AA there was a reduction in binding activity in 0·2 M NaCl when compared to 0·1 M NaCl whereas wild-type pTP bound DNA with comparable efficiency at both NaCl concentrations. Although stability of the pTPDNA interaction to increasing concentrations of NaCl may not be a true measure of binding affinity, it is expected that mutants which retain DNA binding activity at the higher NaCl concentration have a greater affinity for DNA. These results are summarized in Table 2
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Discussion |
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All of the mutants generated are compromised to some extent in their ability to interact with Adpol and DNA. Of the 18 mutants tested 13 were severely compromised for initiation activity and all of the mutants displayed a reduced ability to participate in the initiation reaction. The data are summarized in Table 2.
Mutants such as 137Y/A, 139R/A, aa 183207 del, 437438EE/AA and 529R/A bind both Adpol and DNA reasonably well, but are severely compromised in their ability to participate in the initiation of DNA replication. These mutants are presumably incorporated into the preinitiation complex but fail to act as a primer onto which Adpol can transfer dCMP. Possible explanations for this behaviour are that the hydroxyl group of serine-580 is not precisely aligned in the active site of Adpol and as such fails to prime DNA replication. Alternatively, pTP may have to undergo a conformational change during the initiation process and the mutants may be defective in this step.
Mutant 356357RE/AA shows the most initiation activity after wild-type pTP. This mutant binds DNA well at 0·2 M NaCl and also binds to Adpol. Two mutants which also display initiation activity are 360R/A and 589Q/A. Both mutants bind DNA reasonably well, although 589Q/A binds to Adpol much more strongly than 360R/A does. The strength of pTP binding to Adpol does not seem to be a major determinant in initiation activity. Mutants 137Y/A, 139R/A, 363T/A, 384385RR/AA, 388389RR/AA and 529R/A all bind to Adpol reasonably well but all show barely detectable or no initiation. Conversely, mutants 386387RR/AA and 598599DS/AA display some of the weakest DNA binding at 0·2 M NaCl but are the next most active for initiation after 356357RE/AA, 360R/A and 589Q/A. It can therefore be seen that the initiation process is controlled by more subtle effects than just the strength of interaction with members of the complex. It is expected that small changes in these interactions, possibly brought about by conformational changes induced in pTP have a major effect on activity by altering the position of serine-580, the catalytic serine.
A number of the mutants in the central portion of pTP have compromised DNA binding activity, indicating that this region of the molecule is involved in interaction with DNA. The N-terminal deletions aa 1175 del (iTP) and aa 1349 del (TP) are compromised for DNA binding in 0·2 M NaCl. This is consistent with previous work (Webster et al., 1997b ) which demonstrated that TP and iTP generated in situ by the Ad2 protease were defective for DNA binding. On the basis of UV cross-linking experiments it was previously demonstrated that the N-terminal region of pTP was in contact with DNA (Webster et al., 1997a
). However, mutants in the N-terminal region (137Y/A and 139R/A) retain DNA binding activity, indicating that these particular residues are not directly involved in interaction with DNA. In addition, it was demonstrated (Webster et al., 1997a
) that antibodies binding to other regions of the molecule disrupted the interaction with the DNA, suggesting that regions of pTP other than the N-terminal portion of the molecule are required for efficient DNA binding. This is supported by the present mutational analysis.
In vivo, pTP exists in a stable complex with Adpol and high concentrations of denaturant (1·7 M urea) are required to separate the subunits. The Adpol binding assay shows that there are multiple regions of the pTP molecule which appear to bind to Adpol. This is consistent with the partial proteolysis experiments (Webster et al., 1997a ) which showed Adpol protecting a region between the iTP and TP cleavage sites, the TP cleavage site itself and a portion of the C terminus. Here, the residues in this region involved in the interaction may be defined more specifically, as mutants 520D/A and 598599DS/AA are very poor at binding Adpol whereas mutants 529R/A and 589Q/A are relatively efficient at binding Adpol. Thus, it seems likely that a large proportion of the pTP surface contributes to the interface with Adpol.
It was predicted that the aa 183207 region might form an exposed loop. Ad4 pTP lacks this region and so it was thought that the mutant with a deletion in this region might mimic Ad4 pTP. However, the deletion mutant was found to initiate DNA replication very inefficiently suggesting that the putative loop region is important for pTP initiation activity. Whether Ad4 pTP manages to circumvent the requirement for this loop, through alterations in other parts of its structure remains unclear.
The aa 1175 (iTP) and aa 1349 (TP) deletions are both unable to initiate. This contrasts with data obtained using iTP produced by in situ digestion with Ad2 protease, where iTP was shown to be efficient at initiation (Webster et al., 1997b ). However, in this experiment it was shown that the cleaved N-terminal portion of pTP remained associated with iTP in that both portions could be co-immunoprecipitated with Adpol. The genetically generated iTP did not bind Adpol which suggests that the N-terminal and iTP portions of pTP need to be associated for Adpol binding and initiation to occur. TP produced by Ad2 protease digestion does not remain associated with the cleaved N-terminal portions and does not bind Adpol or support initiation. These results are in agreement with those obtained using the aa 1349 deletion (TP).
It was noted that the aa 1349 deletion (TP), although it contained the NLS (Zhao & Padmanabhan, 1988 ), was exclusively cytoplasmic. This may highlight a difference in the NLS requirements of insect cells. On the other hand, it may suggest that the NLS is not surface exposed in the baculovirus-expressed TP. Thus, an individual TP molecule may be adopting a conformation different to that adopted by the TP portion of pTP. The mutants 384385RR/AA, 386387RR/AA and 388389RR/AA are still transported to the nucleus although they contain double mutations of arginines to alanines within the NLS. It would, therefore, seem likely that some of the arginines in the NLS are redundant, as predicted from the sequence alignment which shows variations in the number and position of conserved arginines in the more distantly related adenovirus serotypes.
The mutants analysed in this study shed light on the requirements of pTP in different aspects of adenovirus replication and identify regions of pTP associated with these activities.
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Acknowledgments |
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References |
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Received 27 March 2001;
accepted 10 May 2001.