Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures
© Mikhailov and Rohrmann; licensee BioMed Central Ltd. 2002
Received: 21 August 2002
Accepted: 26 September 2002
Published: 26 September 2002
Baculovirus genomes encode a gene called very late expression factor 1 (VLF-1) that is a member of the integrase (Int) family of proteins. In this report we describe the binding properties of purified Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV) VLF-1 to a number of different DNA structures including homologous regions. In addition, its enzymatic activity was examined.
VLF-1 was expressed in a recombinant baculovirus as a fusion with both HA and HIS6 tags and its binding activity to different DNA structures was tested. No binding was evident to single and double strand structures, very low binding was observed to Y-forks, more binding was observed to three-way junctions, whereas cruciform structures showed high levels of binding. VLF-1 binding was affected by divalent cations; optimal binding to three-way junctions and cruciforms was 2 and 0 mM MgCl2, respectively. Homologous region (hr) sequences was also examined including oligomers designed to expose the hr palindrome as a hairpin, linear double strand, or H-shaped structure. Efficient binding was observed to the hairpin and H-shaped structure. No topoisomerase or endonuclease activity was detected. Sedimentation analysis indicated that *VLF-1 is present as a monomer.
An HA- and HIS-tagged version of AcMNPV VLF-1 showed structure-dependent binding to DNA substrates with the highest binding affinity to cruciform DNA. These results are consistent with the involvement of VLF-1 in the processing of branched DNA molecules at the late stages of viral genome replication. We were unable to detect enzymatic activity associated with these complexes.
Baculoviruses contain double-stranded, circular, covalently closed DNA genomes of 100–180 kb [1, 2]. A distinctive feature of their genomes is the presence of homologous regions (hrs) located at a number of positions. In the best characterized baculovirus, Autographa californica multiple capsid nucleopolyhedrovirus (AcMNPV), the hr repeat units contain about 70-bp with an imperfect 30-bp palindrome located near the center, and are repeated two to eight times at each of eight locations around the genome. Hrs have been implicated both as transcriptional enhancers and origins of DNA replication for a number of baculoviruses [3–9]. Hr-containing plasmid DNA transfected into AcMNPV-infected cells appears to replicate as high molecular weight concatemers containing a number of copies of the plasmid . Evidence also suggests that the AcMNPV genome may replicate via a rolling circle mechanism that results in large concatemeric DNA intermediates that are resolved into genome length sizes .
All baculovirus genomes sequenced to date encode a gene called very late expression factor 1 (VLF-1) that was originally identified using an AcMNPV temperature sensitive mutant that failed to produce occlusion bodies at the non permissive temperature . VLF-1 is a member of the integrase (Int) family of proteins which are a large group of enzymes that includes many site-specific DNA recombinases . They are found in a variety of organisms including viruses where they are involved in the integration and excision of viral genomes and decatenation of newly replicated viral chromosomes. The baculovirus VLF-1 gene has been demonstrated to be required for very late gene expression and evidence suggests that it binds to the polyhedrin promoter region . Therefore, it is likely to be involved in stimulating the baculovirus RNA polymerase to express high levels of polyhedrin which is required for occlusion body formation. The AcMNPV vlf-1 gene may also encode an essential function associated with a putative integrase/resolvase activity  and this activity may be involved in the processing of concatemeric replicative intermediates into monomeric DNAs. Members of the int family encode four conserved amino acids (R, H, R, Y) that are involved in the active site and catalyze DNA rearrangements . Baculovirus VLF-1 sequences align with three of these amino acids (R, R and Y but not H). Mutagenesis of the tyrosine residue, that forms a covalent bond with DNA during recombination reactions in int homologs, resulted in the inability of mutant AcMNPV to produce infectious virions, but did not affect activation of very late promoters . In addition, VLF-1 has been found to be associated with viral nucleocapsids , which indicates that it could be involved in genome processing concomitantly with DNA packaging. Therefore, it has been suggested that AcMNPV VLF-1 may be involved in both transcriptional activation of very late genes and in the processing of viral DNA.
In this report we describe the binding properties of purified AcMNPV VLF-1 to a number of different DNA structures which are predicted to mimic the late replicative and recombination intermediates including those containing hr sequences. In addition, the ability of VLF-1 to cleave such structures was also examined.
Expression and purification of HAHIS-VLF-1 (*VLF-1)
Binding of *VLF-1 to DNA structures
Binding of *VLF-1 to homologous regions
Testing for DNA cleavage and topoisomerase activity
It has been found that modification of the N-terminus of phage λ integrase affected protein-protein interactions . Because *VLF-1 was tagged with both HIS6 and HA sequences, we considered that these changes at the N-terminus may have inhibited the enzymatic or binding activity of VLF-1. In order to investigate this possibility, we purified VLF-1 from a virus called vpolhHisVlf1 in which a HIS-tagged version of VLF-1 under the control of the polyhedrin promoter was substituted for the wt VLF-1 gene. This virus is replication competent as evidenced by its production of budded virus at wt levels  indicating that if VLF-1 is involved in the production of mature viral genomes, the genome is correctly processed. We purified this version of VLF-1 and tested it in the endonuclease assay using the cruciform structures, but again, no activity was detected. In addition, when tested in a topoisomerase assay with superhelical plasmid DNA, no activity could be correlated with this protein.
Sedimentation analysis of *VLF-1 in glycerol gradients
VLF-1 has homology to members of the phage λ integrase family of tyrosine recombinases and evidence suggests that the VLF-1 plays two independent roles in baculovirus infection cycle. These include a role as a transcription factor for the high level expression of very late genes polyhedrin and p10 [13, 21] which is independent of the conserved tyrosine residue. Mutagenesis of the conserved tyrosine, although not affecting very late gene expression, results in an inability of the mutant to produce viable virus. This suggests that VLF-1 may also be involved in genome replication . In particular, it may be involved in the resolution of complex branched concatenated structures that may be produced during genome replication [7, 10]. In agreement with this theory, the purified HA- and HIS-tagged VLF-1 showed a structure-dependent binding to DNA substrates. Our data indicates that VLF-1 has the highest binding affinity for cruciform structures. These structures would likely mimic recombination intermediates (Fig. 2) which is consistent with VLF-1 being involved in resolution of these structures. *VLF-1 was also shown to bind two different hr-containing substrates that would form the hairpin-like and H-shaped structures (Fig. 5). Hrs (homologous regions) appear to be a common feature of many baculoviruses and vary from imperfect palindromes to imperfect direct repeats in different viruses . Although they appear to act as enhancers of early gene transcription and replication origins (reviewed in ), they could also act as signals for genome processing. However, in our experiments when the hr sequences were inserted into DNA substrates, they did not appear to specifically affect *VLF-1 binding.
Although we clearly showed that VLF-1 is capable of binding to branched DNA structures consistent with a role in genome processing, we were unable to detect enzymatic activity associated with these complexes. If VLF-1 is involved in genome processing as we suspect, there are a number of reasons that might explain this result. It is possible that it might require another viral or host protein for activity. Phage λ integrase requires two host (E. coli) encoded proteins in order to carry out recombination in vitro . It may be site-specific and it is possible that we have not employed a compatible DNA sequence in our assays. It could require superhelicity of its DNA substrate in a manner similar to eukaryotic topoisomerase I which has a preference for supercoiled DNA ( and reviewed in ). It is also possible that we have not identified the correct conditions for its enzymatic activity or it may become inactivated during our purification protocol.
An HA- and HIS-tagged version of AcMNPV VLF-1 showed structure-dependent binding to DNA substrates with the highest binding affinity for cruciform DNA which mimics a structure common to recombination intermediates. These results are consistent with the involvement of VLF-1 in the processing of branched DNA molecules at the late stages of viral genome replication. Although we showed the VLF-1 is capable of binding to branched DNA structures, we were unable to find enzymatic activity associated with these complexes.
Chemicals and enzymes
Radiolabeled nucleotide [γ-32P]ATP was from Perkin-Elmer, and T4 polynucleotide kinase was from New England Biolabs, bacteriophage T4 endonuclease VII was the kind gift of Dr. B. Kemper .
Spodoptera frugiperda 9 (Sf9) cells were cultured in Sf900II serum-free media (Invitrogen, Inc.), penicillin G (50 units/ml), streptomycin (50 μg/ml, Whittaker Bioproducts), and fungizone (amphotericin B, 375 ng/ml, Flow Laboratories) as previously described .
Transfer plasmids and virus construction
Recombinant baculovirus vfbHAHISVlf-1 for overexpression of an AcMNPV His6-tagged VLF-1 (orf77) under the control of the polyhedrin promoter was produced as previously described . The transfer plasmids were constructed as follows: The plasmid pHSEpiHisVLF-1  was a gift from Lois Miller. It was digested with BglII, treated with calf intestinal phosphatase and modified using two oligonucleotides that were phosphorylated by treatment with polynucleotide kinase and ATP, gel purified, annealed , and inserted into the BglII site. The oligo names and sequences are:
5bamhahisc – GATCGGGATC CGACCATGAG CTCCCGATAC CCATACGACG TCCCAGATTA CGCCCGGCAT CATCATCATC ATCATCACC
3bamhahisc – GATCGGTGAT GATGATGATG ATGCCGGGCG TAATCTGGGA CGTCGTATGG GTATCGGGAG CTCATGGTCG GATCCC.
This resulted in the creation of an ATG translation start site followed by a HA epitope and a HIS tag upstream of the ORF, but downstream of a new BamHI site (see ). Clones were screened using BamHI digestion (the correct orientation is about 70 bp smaller than the reverse orientation), and presumptive correct plasmids were sequenced to confirm the correct orientation of the insert. The primer used for confirming the sequence of the altered region was called AcVLF250 and has the coordinates 64681–64700 (TCCAACGAGTACGACATGTC) . The ORF was then removed by digestion with BamHI and NotI, gel purified, and inserted into a pFastbac1 vector (Gibco-BRL) digested with the same enzymes and gel purified. This plasmid was then used to produce the recombinant baculovirus vfbHAHISVlf-1 using the Bac-to-Bac Baculovirus Expression system (Gibco-BRL) following the manufacturer's instructions.
The virus vpolhisvlf-1  was a gift from Dr. L. Passarelli. It contains the VLF-1 gene expressed from the polyhedrin promoter and is His tagged and produces wt levels of budded virions. However, in contrast to vfbHAHISVlf-1, the VLF-1 that it expresses lacks an HA tag.
All oligomers were synthesized and PAGE purified by QIAGEN Operon, Inc. The oligonucleotides to produce different structures are identical to those used by . They include the following (61–63)-mers:
The oligonucleotide 4X12-2 was 5' end-labeled using T4 polynucleotide kinase and [γ-32P]ATP. For the various DNA conformations the labeled oligonucleotide was annealed with a 1.3-fold excess of unlabeled oligonucleotides in the following combinations: single-stranded DNA (substrate I), none; double-stranded DNA, 4X12-5 (substrate II); for a pseudo-Y fork 4X12-1(substrate III); for a three-way junction, 4X12-1 plus 4X12-3 (substrate IV); for a cruciform, 4X12-1 plus 4X12-3 plus 4X12-4 (substrate V). The cruciforms (substrates V) with any one of four oligonucleotides, 4X12-1, 4X12-2, 4X12-3 or 4X12-4, labeled with 32P at the 5' end were prepared in the same manner.
The oligonucleotides that were used to examine binding to homologous repeat (hr) sequences are:
80-mer v-hr1: 5'GGGGTTTGTTTTTCAAAACTAAACTCGCTTTACGAGTAGAATTCTACTTGTAACGCACAATCAAGGGATGATGTCAGGGG3'
80-mer v-hr2: 5'CCCCTGACATCATCCCTTGATTGTGCGTTACAAGTAGAATTCTACTCGTAAAGCGAGTTTAGTTTTGAAAAACAAACCCC3'
50-mer v-hr3: 5'CCCCTGACATCATCCCTTGATTGTGAGTTTAGTTTTGAAAAACAAACCCC
The oligonucleotide v-hr1 was labeled with 32P at 5' end and annealed with v-hr2 to form the linear double-stranded hr containing substrate (substrate VI), or with v-hr3 to form the hairpin hr DNA (substrate VII) and double forked hr DNA (substrate VIII).
Purification of HAHIS-VLF-1 (*VLF-1)
Sf9 cells at a density of 1.5 × 106/ ml in shaker flasks were infected with the recombinant baculovirus vfbHAHISVlf-1 at a multiplicity of infection (MOI) of about 4, and incubated with shaking for 72 h at 28°C. *VLF-1 (HAHIS-VLF-1) was purified routinely from 100-ml cultures of infected cells by liquid chromatography sequentially on Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen), DEAE-Toyopearl 650 (TosoHaas), and heparin-Sepharose CL-6B (Amersham Pharmacia Biotech) columns. The infected cells were pelleted by centrifugation for 5 min at 500 × g and resuspended in 8 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.5), 200 mM KCl, 1% Nonidet P-40, 5 mM 2-mercaptoethanol and a set of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, 5 μg of leupeptin per ml, 5 μg of aprotinin per ml, 2 μg of E64 per ml). After extraction for 15 min at 4°C on a rotating shaker, the preparation was clarified by centrifuged at 30,000 × g for 30 min. The supernatant was loaded onto a Ni-NTA-agarose column (0.8 ml), equilibrated with buffer A (20 mM Tris-HCl [pH 8.5], 0.5 M KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol, 20 mM imidazole). The column was washed successively with 10 ml of buffer A, 2.5 ml of buffer B (20 mM Tris-HCl [pH 8.5], 1 M KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol, 20 mM imidazole), 1.5 ml of buffer A, and finally with 2 ml of buffer C (20 mM Tris-HCl [pH 8.5], 75 mM KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol) containing 20 mM imidazole.
Protein was eluted from the column with 4 ml of buffer C containing 150 mM imidazole. The sample was loaded at a rate of 4 ml per h onto a DEAE-Toyopearl column (0.5 by 2.5 cm) equilibrated with buffer D (10 mM Tris-HCl [pH 7.5], 20% [vol/vol] glycerol, 1 mM dithiothreitol [DTT], 1 mM EDTA) containing 75 mM KCl. The column was washed successively with 1 ml of buffer D containing 75 mM KCl, 3 ml of buffer D containing 100 mM KCl, and protein was then eluted with 3 ml of the same buffer containing 240 mM KCl. The sample was loaded on a 0.5-ml column of heparin-Sepharose equilibrated with buffer D containing 240 mM KCl. The column was washed with 2 ml of buffer D containing 240 mM KCl, 3 ml of the same buffer containing 280 mM KCl, and *VLF-1 was then eluted with sequential 1-ml portions of buffer D containing KCl in final concentrations of 0.31, 0.35, 0.40, and 0.5 M. Proteins from each fraction were analyzed by SDS-10% PAGE followed by staining with Coomassie brilliant blue. The fractions collected at 0.35 and 0.4 M KCl were combined or dialyzed separately against buffer E (10 mM Tris-HCl [pH 7.5], 50% [vol/vol] glycerol, 1 mM DTT, 0.2 mM EDTA), and stored at -20°C for periods of 1 to 2 months or at -80°C for long-term storage. Protein concentrations were determined by SDS-PAGE followed by optical densitometry of the gel stained with Coomassie brilliant blue. Bovine serum albumin (BSA) loaded in different amounts on separate lanes of the same gel was used for generation of the calibration curve.
PAGE and Western blotting
SDS-(10 or 12%) PAGE was performed as described by Laemmli . Gels were either fixed and stained, or electrophoretically transferred to PVDF-Plus transfer membranes (Micron Separations Inc) using a Trans-blot SD semidry transfer cell (Bio-Rad) according to the manufacturer's guidelines. Western blots were probed with 1:1,000 dilution of monoclonal antibody HA.11 (BAbCO), washed, incubated with a 1:3,000 dilution of goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad Laboratories), and developed using ECL detection reagents (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Assays for endonuclease activity of *VLF-1
Reaction mixtures (10 μl) contained 0.004 pmol of substrate V (cruciform DNA) with one of four 62-mers labeled with 32P at 5' end, 20 mM Tris-HCl (pH 8.0), 1.5 mM MgCl2, 100 μg of BSA per ml, and 2 mM DTT. In some experiments, the substrate V was replaced with a mixture of hairpin hr DNA (substrate VII) and double forked (double Y-form) hr DNA (substrate VIII) (total 0.008 pmol), 25 mM Tris-HCl buffer was used at pH 7.5 or pH 8.8, and the mixtures contained no monovalent salt or 100 mM KCl. Purified *VLF-1 (40 or 80 ng in 3 μl of buffer D) was added to a final volume of 10 μl. After incubation for 1 h at 37°C, reactions were terminated by chilling on ice and adding 7 μl of stop solution (95% formamide, 20 mM EDTA, 0.05% each bromophenol blue and xylene cyanol). After heating for 10 min at 85°C, a 6-μl portion of each reaction was loaded onto a 15% polyacrylamide-8 M urea slab gel (17 by 14.7 by 0.08 cm). Electrophoresis was performed in TBE (Tris-borate-EDTA) buffer at 450 V for 1.5 to 2 h until the bromophenol blue migrated 3 cm above the bottom of the gel. The gel was transferred onto a polymer support and exposed to X-ray film at -80°C with an intensifying screen.
Topoisomerase activity of *VLF-1 was assayed as previously described for the baculovirus replication factor LEF-1 .
Assay conditions for bacteriophage T4 endonuclease VII
Reaction mixtures containing 0.004 pmol of 32P-labeled oligonucleotide probe (substrates I to V), 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 100 μg of BSA per ml, and 2 mM DTT were assembled on ice. Bacteriophage T4 endonuclease VII (20 units in 3 μl of buffer D) (the gift of Prof. Börries Kemper) was added to a final volume of 10 μl. After incubation for 20 min at 37°C, reactions were terminated by the addition of EDTA to 15 mM and SDS to 0.5%, and the samples were treated with proteinase K (100 μg/ml) for 30 min at 37°C. Half of each reaction mixture was loaded onto a 6% polyacrylamide (acrylamide-bisacrylamide, 60:1) slab gel (6 by 10 by 0.075 cm) prepared in a buffer containing 20 mM HEPES (pH 8.0) and 0.1 mM EDTA. Electrophoresis was performed at 80 V in the same buffer until the bromophenol blue migrated 12 mm above the bottom of the gel. The gel was transferred onto Whatman DE81 paper, dried under vacuum, and then exposed to X-ray film at -80°C with an intensifying screen
Electrophoretical mobility shift assay
Reaction mixtures (10 μl) contained 0.004 pmol of 32P-labeled oligonucleotide probe (substrates I to VIII), 20 mM HEPES (pH 8.0), 50 mM KCl, 100 μg of BSA per ml, and 2 mM DTT. Various amounts of the purified *VLF-1 were added in 3 μl of buffer E. The assembled reaction mixtures were incubated for 20 min on 23°C. For fixation, 0.5 μl of 10% glutaraldehyde was then added to each mixture, and the incubation was continued for 30 min. Half of each reaction mixture was loaded onto a 6% polyacrylamide (acrylamide-bisacrylamide, 60:1) slab gel (6 by 10 by 0.075 cm) prepared in a buffer containing 20 mM HEPES (pH 8.0) and 0.1 mM EDTA. Electrophoresis was performed at 80 V in the same buffer until the bromophenol blue migrated 5 to 10 mm above the bottom of the gel. The gel was transferred onto Whatman DE81 paper, dried under vacuum, and then exposed to X-ray film at -80°C with an intensifying screen.
Sedimentation in glycerol gradients
Linear 15 to 30% glycerol gradients were prepared in buffer F (0.4 M KCl, 10 mM Tris-HCl [pH 7.5], 1 mM DTT, 1 mM EDTA). Purified *VLF-1 (10 μg in 60 μl), after dialysis against buffer F containing 5% glycerol, was layered over 4.8 ml of a the gradient prepared in nitrocellulose tubes. Ovalbumin (45 kDa, 3.55 S) and BSA (66 kDa, 4.3 S) (0.2 mg of each) were centrifuged in individual tubes as sedimentation standards. After centrifugation in the SW 50.1(Beckman) rotor at 48,000 rpm and 4°C for 22 h, the gradients were fractionated from the bottom with a peristaltic pump. The presence of *VLF1 was monitored by SDS-10% PAGE followed by silver staining.
We thank Dr. Kazuhiro Okano for participation in some experiments. The gift of reagents from Drs. Lois Miller, Lorena Passarelli, and Boris Kemper are gratefully acknowledged. This research was supported by grants from the NIH (GM9982536) to G.F.R. and from the Russian Foundation for Basic Research (00-04-49237) to V.S.M.. This is Technical Report No. 11933 from the Oregon State University Agricultural Experiment Station.
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