Biomolecular Sciences Building, University of St Andrews, North Haugh, St Andrews KY16 9ST, UK
Correspondence
G. D. Kemp
gdk{at}st-andrews.ac.uk
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ABSTRACT |
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Present address: Institut Jacques Monod, CNRS, Tour 4344, 2 Place Jussieu, 75005 Paris, France.
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INTRODUCTION |
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One distinctive characteristic of adenain is that its activity is regulated by the formation of a disulphide-linked heterodimer with an 11 aa peptide (GVQSLKRRRCF) derived from the C terminus of another adenoviral protein, pVI, and termed pVI-CT (Mangel et al., 1993; Webster et al., 1993
). It has been suggested that the disulphide-linked pVI-CTadenain dimer is required for activation and that a form of thioldisulphide interchange results in formation of the active heterodimer, involving C104 of adenain (Webster et al., 1993
).
Ding et al. (1996) described adenain as an ovoid structure with two mini-domains, one characterized by a four-stranded sheet and the other containing three major
-helices. The active-site histidine (H54) lies at the end of one of the
-strands, whilst the nucleophilic cysteine (C122) is part of one of the
-helices. G1 and V2 of pVI-CT fit into a pocket that is formed between two of the major
-helices, whereas aa 410 form an additional strand to the
-sheet, indicating that activation may be a result of tying together the two mini-domains and creating and stabilizing the active conformation (Cabrita et al., 1997
).
Recently, Wodrich et al. (2003) drew attention to the fact that pVI contains two nuclear export signals (NESs) and two nuclear localization signals (NLSs). One NLS is located in pVI-CT and one NES straddles the boundary between pVI-CT and the remainder of pVI (iVI). These authors demonstrated that pVI is capable of transporting the adenovirus hexon protein to the nucleus and further suggested that, as part of that role, pVI is capable of shuttling between the cytoplasm and the nucleus.
Alignment of the pVI-CT sequences from human adenoviruses shows that aa 69 are completely conserved in a distinctive basic motif, KRRR (Fig. 1). The aim of the work described here was to determine the influence of this motif on the function of the adenain system. The data presented show that these residues are much less important than G1, V2, C10 and F11 in the binding of pVI-CT to the protease and for the activity of the heterodimer. These findings are in accord with those of Baniecki et al. (2001)
, which were obtained by using a different binding assay. This motif does, however, constitute one of two potential NLSs in pVI that were reported by Wodrich et al. (2003)
. The data presented here confirm and extend the observations of Wodrich et al. (2003)
by defining the minimum sequence that is necessary by mutational analysis. We also demonstrated that, whilst both NLSs were required for nuclear localization of pVI, the KRRR motif was capable of targeting other proteins to the nucleus on its own. Furthermore, it caused an intranuclear distribution into distinct foci, which is characteristic of the pVI distribution that is observed during adenovirus infection. We have also reported the use of this motif in the construction of fluorescent substrates for adenain that are capable of demonstrating the appearance of adenain activity in the cell nucleus following infection.
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METHODS |
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Expression and purification of adenain.
Adenain was expressed in and purified from Escherichia coli BL21(DE3) as described previously (Anderson, 1990; Grierson et al., 1994
). Protein and peptide concentrations were determined as described previously (Cabrita et al., 1997
).
Non-denaturing, non-reducing, native gel electrophoresis.
The native gel electrophoresis for basic proteins was a method adapted from that of Goldenberg (1989). To obtain native conditions for adenain and pVI-CT binding, riboflavin was used with tetramethylethylenediamine for photopolymerization in the absence of SDS. In a typical binding assay, 4 µl containing 400 ng adenain and 500 ng lysozyme in 50 mM Tris/HCl (pH 8) was mixed with 5 µl of varying concentrations (1 mM to 4 µM in 50 mM Tris/HCl, pH 8) of pVI-CT or its variants and 12·5 µl 50 mM Tris/HCl, 10 mM EDTA, 2 mM 2-mercaptoethanol. After 5 min at room temperature, samples were mixed with 10 µl 0·8 % methyl green, 20 % glycerol; 20 µl from each mixture was applied to a 15·4 % (w/v) polyacrylamide gel. Following electrophoresis for 40 min at 180 V, gels were stained and destained as described previously (Webster et al., 1993
). Gels were scanned by using a Canoscan FB 636U scanner and densitometric analysis of the bands was performed by using the public domain NIH Image program (developed at the US National Institutes of Health; http://rsb.info.nih.gov/nih-image/).
In the absence of activating peptide, adenain migrates as a diffuse band; the presence of lysozyme was used to normalize total adenain concentration. In all gels, one reference lane contained wild-type pVI-CT in a 300-fold molar excess over adenain. Under these conditions, all of the adenain is found in the heterodimer. The proportion of adenain complexed with activating peptide (and thus the amount of bound activating peptide) in the other lanes was determined by comparison with this reference lane. This process assumes that adenain and pVI-CT form a 1 : 1 complex, as indicated by the structural studies of Ding et al. (1996).
Activity assays.
The Km and kcat values for protease and peptide mutants were obtained from activity assays with the fluorogenic substrate z-Leu-Arg-Gly-Gly-AMC (z-LRGG-AMC; Bachem). Activity assays were performed in assay buffer (50 mM Tris/HCl, 10 mM EDTA, pH 8, with 2 mM 2-mercaptoethanol) with 26 nM adenain and 2 µM peptide. The assay mixtures were pre-incubated at 37 °C for 5 min before the addition of the fluorogenic substrate from a 50 mM stock solution in DMSO. Increase in fluorescence was measured by using a Perkin-Elmer LSB50 luminescence spectrometer at excitation wavelength 370 nm and emission wavelength 460 nm. Each experiment was performed in triplicate. In order to determine kcat values, the concentration of activated protease was calculated as described by Baniecki et al. (2001) from Kd values obtained by native gel electrophoresis.
In experiments to determine Kd from the kinetic constants, 26 nM purified adenain and varying concentrations of peptide (0·33 µM) were incubated in assay buffer prior to the addition of 62·5 µM substrate. Assays were performed at 37 °C. For Km and kcat determinations, the peptide concentration was kept at 2 µM and the substrate concentration was altered from 15·6 to 200 µM. Otherwise, the experiments to determine Kd by this approach were conducted in an identical manner to the determination of Km and kcat.
Cell culture, transfections and infections.
HeLa cells were used in transient transfections and infections by human adenovirus type 2 (Ad2). The cells were maintained at 37 °C in 5 % CO2 in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10 % fetal calf serum (Gibco). Cells were transfected by using FuGene transfection reagent (Roche) according to the manufacturer's instructions. Cells were fixed 1624 h after initial transfection with 3 % paraformaldehyde in PBS. For microscopy, cells were grown on glass cover slips, which were mounted on to slides with 90 % glycerol/10 % PBS containing DAPI. For the pyruvate kinase (PK) constructs, 9E10CD antibody, which is specific for PK (a gift from C. Dargement, Institut Curie-CNRS, Paris), was used, together with secondary antibody conjugated to fluorescein isothiocyanate (FITC; Oxford Biotechnology). For the labelling of pVI/iVI/VI in infected cells, a rabbit polyclonal anti-pVI antibody, raised against aa 94170 of pVI of Ad2, was used (a gift from W. C. Russell, University of St Andrews, UK), together with FITC-conjugated anti-rabbit Ig.
Virus propagation.
The preparation and purification of Ad2 was performed as described by Russell & Blair (1977). Viral infections were performed at an m.o.i. of 510.
Fluorescence microscopy.
Fluorescent images were acquired by using a DeltaVision restoration microscope (Applied Precision) and analysed by using softWoRx software. Indirect immunofluorescence was performed as reported previously (Rodriguez et al., 2001). Images were acquired by using a Nikon Microphot-FXA microscope and a 100x oil-immersion lens.
Cytoplasmic and nuclear fractionation of transfected cells.
To obtain separate cytoplasmic and nuclear extracts from transfected cells, cells grown on six-well plates were transfected by using FuGene. At 24 h after initial transfection, cells were removed by using a cell scraper and resuspended in 200 µl 10 mM HEPES (pH 8), 50 mM NaCl, 0·5 M sucrose, 1 mM EDTA, 0·5 mM spermidine, 0·15 mM spermine and 0·2 % Triton X-100. The lysate was left on ice for 6 min and centrifuged at 6500 r.p.m. by using a Sigma 3K10 centrifugator for 3 min at 4 °C. The resulting supernatant was stored at 70 °C as the cytoplasmic fraction. The pellet was washed with 200 µl 50 mM NaCl, 10 mM HEPES, 25 % glycerol, 0·1 mM EDTA, 0·5 mM spermidine, 0·15 mM spermine and centrifuged at 6500 r.p.m. for 3 min at 4 °C, after which the supernatant was discarded. The pellet was resuspended carefully in 100 µl 350 mM NaCl, 10 mM HEPES, 25 % glycerol, 0·1 mM EDTA, 0·5 mM spermidine and 0·15 mM spermine and left on ice for 30 min with regular mixing. The lysate was centrifuged at 6500 r.p.m. for 20 min at 4 °C and the supernatant was stored at 70 °C as the nuclear extract until further analysis. All buffers contained a cocktail of protease inhibitors (Roche), added immediately before use.
SDS-PAGE and Western blotting.
The nuclear and cytoplasmic fractions were subject to 15 % SDS-PAGE followed by transfer to nitrocellulose membrane by Western blotting, as described previously (Grierson et al., 1994). To detect expression of pVIEGFP and mutants (see below), a rabbit anti-GFP antibody (Roche) was used.
Plasmid construction.
For all plasmid constructs, the oligonucleotides for PCR were purchased from Oswel DNA Services. The constructs generated were sequenced at the University of St Andrews DNA sequencing unit. Vent DNA polymerase (New England Biolabs) was used for the PCRs, which were conducted according to the manufacturer's instructions.
Construction of pVI and mutants in pEGFP-C1.
pVI cDNA was amplified by PCR from Ad2 and the PCR-generated mutants were inserted at the C terminus of the pEGFP-C1 and pDsRED2-C1 (containing red fluorescent protein) plasmids (Clontech), using the EcoRI/BamHI restriction sites (underlined below). The oligonucleotides used for amplification of the C-terminal mutant of pVIEGFP (mutation of KRRR to four alanines) were (1) 5'- GCTCAAGCTTCGAATTCCATGGAAGAC-3' (N terminus of pVI is shown in bold) and (2) 5'-CGGTGGATCCTTAGAAGCATGCTGCGGCCGCCAGGGATTGCAC-3' (introduced mutations are shown in bold). To obtain the middle mutant (MM) of pVIEGFP, the KRPRP sequence was mutated to five alanines by using oligonucleotide (1) together with oligonucleotide (3) (5'-CCTGTCGGCCGCCGCCGCCGCTTCGCCACGCCC-3'). The resulting fragment was used together with the C-terminal pVIEGFP oligonucleotide (4) (5'-CGGTGGATCCTTAGAAGCATCGTCG-3') for a second PCR to obtain the full-length insert. The double mutant (DM) of pVI was obtained by using the MM as a template for the PCR with oligonucleotides (1) and (2). Truncated forms of pVI were synthesized as follows. The 34250 aa truncated pVI protein was generated by using oligonucleotide (4) together with oligonucleotide (5) (5'-GCTTCGAATTCTGCCTTCAGCTGGGGCTCG-3'). Similarly, the intermediate form of pVI (iVI; aa 1239) was generated by using oligonucleotides (1) and (6) (5'-GGAGGATCCTTACAGACCCACGATGCTG-3'). The mature VI (aa 34239) was synthesized by combining oligonucleotides (5) and (6).
Construction of PK fusion proteins.
The KRRR sequence and single alanine mutants thereof were expressed in plasmid pcDNA-PK. Synthetic complementary oligonucleotides were annealed and cloned into the BamHI/XbaI sites at the C terminus of PK. The sequences of the sense oligonucleotides [restriction sites underlined, mutation(s) in bold] were: PKKKRKV (SV40 large T antigen NLS), 5'-GATCCGCTCCAAAGAAGAAGCGCAAGGTGGAATAGT-3'; KRRRCF, 5'-GATCCTCTCTTAAACGTCGTCGTTGTTTTTAGG-3'; KARACF, 5'-GATCCTCTCTTAAAGCTCGTGCTTGTTTTTAGT-3'; AARACF, 5'-GATCCTCTCTTGCTGCTCGTGCTTGTTTTTAGT-3'; ARRRCF, 5'-GATCCTCTCTTGCTCGTCGTCGTTGTTTTTAGT-3'; KARRCF, 5'-GATCCTCTCTTAAAGCTCGTCGTTGTTTTTAGG-3'; KRARCF, 5'-GATCCTCTCTTAAACGTGCTCGTTGTTTTTAGG-3'; KRRACF, 5'-GATCCTCTCTTAAACGTCGTGCTTGTTTTTAGG-3'.
Construction of fluorescent substrates based on pVI.
Substrates containing pVI or its derivatives fused to the enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) were prepared as follows. The pcDNA 3.1-based plasmid pdf20 (a gift from P. de Felipe, University of St Andrews, UK) was used as a template for the constructs 20.120.6. The plasmid pdf20, containing the cytomegalovirus promoter, contained EYFP2AECFP2APac inserts (2A, picornavirus 2A protein; Pac, puromycin-N-acetyltransferase). ECFP and EYFP sequences were from Clontech. In this study, 2AECFP2A inserts were removed by XbaI/ApaI digestion followed by insertion of the pVI sequence or C-terminal fragments of it, by using the above restriction sites. To obtain constructs 20.2, 20.4 and 20.6, the ECFP2A fragment was inserted back into the C terminus of the pVI sequence in constructs 20.1, 20.3 and 20.5 by using the ApaI site. The following primers were used for PCRs: 5'-CAAAAGGGCCCACCGCCAGCGGCCGCGAAGCATCGTCGGCGCTTCAGGGA-3' (pVI C-terminal primer for all constructs); 5'-GAAAATCTAGAGGTGGCGGAGAATTCGGTCCGCGATCGATGCGGCCCGTAGCC-3' (for construct 20.1: 34 aa from the C terminus of pVI); 5'-GAAAATCTAGAGGTGGCGGAGAATTCGTGCTGGGCCAGCACACACCTGTAACG-3' (for construct 20.3: 79 aa from the C terminus of pVI); and 5'-GAAAATCTAGAGGTGGCGGAGAATTCATGGAAGACATCAACTTTGCGTCTCTGG (for construct 20.5, full-length pVI). ApaI/XbaI restriction sites in the oligonucleotides are underlined and sequences from pVI are shown in bold. The glycine residues (in italics) were inserted to allow flexibility between the fluorescent proteins and the pVI inserts.
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RESULTS |
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Alignment of pVI-CT sequences from human adenovirus serotypes (Fig. 1) showed that these residues were well-conserved, but that there was also conservation of a distinctive 4 aa basic motif (KRRR) occupying positions 69. The importance of these residues was investigated by using a technique based on gel electrophoresis under non-denaturing conditions (native gel electrophoresis). In the absence of SDS or mercaptoethanol, adenain does not migrate as a distinct band, but rather as a diffuse smear. However, this changes in the presence of pVI-CT and quantification of the distinct band of the proteinpeptide heterodimer provided a measure of the concentration of bound pVI-CT and allowed the determination of binding constants (Fig. 2
).
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The KRRR motif can act as an NLS
To establish whether the KRRR motif could function as an NLS, the wild-type sequence and single alanine mutants thereof were assayed for their ability to confer nuclear accumulation of an otherwise cytoplasmic protein, Myc-tagged PK. The SV40 large T antigen has one of the best-characterized NLSs, the PKKKRKV sequence (Kalderon et al., 1984) and in this study, this sequence, which is capable of directing a cytoplasmic protein to the nucleus, was used as a positive control. For immunofluorescence, the generated PK fusion proteins were detected by using an anti-Myc antibody (9E10).
As illustrated in Fig. 3, in addition to the PKSV40 NLS construct, the PK KRRR construct (the C terminus of pVI-CT) was sufficient to confer nuclear accumulation. PK KRRA and KRAR constructs were also detected in the nucleus, albeit to a lesser extent, especially in the case of KRAR. This sequence has previously been described as an NLS for adenovirus fibre protein. However, in the case of adenovirus fibre experiments, KRAR was also unable to target E. coli
-galactosidase, a cytoplasmic protein, solely to the nucleus (Hong & Engler, 1991
).
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Role of the KRRR motif in localization of pVI
The adenovirus pVI sequence contains two putative NLSs. In addition to the KRRR motif at aa 245248, close to the C terminus of this 250 aa protein, another potential NLS is situated in the middle of the protein, namely the KRPRP motif at aa 131135. This motif is conserved in the pVI sequences from other human serotypes and is also found in the adenovirus E1a protein, and has been proposed to act as an NLS (Lyons et al., 1987).
To assess the relative importance of the KRPRP and KRRR sequences for the nuclear accumulation of pVI, they were mutated individually to alanine residues to obtain MM and C-terminal mutants (CM), respectively. Both putative signal sequences were abolished to obtain the DM. These mutants were cloned and expressed in fluorescent vectors pEGFP-C1 and pDsRED2-C1. In addition, truncated forms of pVI, corresponding to aa 34250, 1239 and 34239, were generated. Two of these mimic the natural cleavage products of adenain, 1239 being the intermediate iVI and 34239 being the mature VI. The localization of these was assessed by fluorescence microscopy and Western blotting analysis of nuclear and cytoplasmic fractions of transfected HeLa cells at 24 h after initial transfection.
Söling et al. (2002) found that the fluorescent protein tag affected the nuclear localization of herpes simplex virus type 1 thymidine kinase. In our study, the pVI protein and its mutants localized in an identical manner, whether expressed as a fusion with EGFP or DsRED2 fluorescent protein (EGFP images only are presented here).
As seen in Fig. 4(a), the CM and MM mutants were found in both the cytoplasm and the nucleus with a punctate distribution. The DM, however, was mainly found in the cytoplasm. None of the mutants was capable of complete nuclear accumulation similar to that observed with the wild-type pVI protein. The synthesized truncated proteins 1239 (iVI) and 34239 (VI) localized similarly, having both lost one of their NLSs. The deletion mutant 34250 was also incapable of complete nuclear accumulation, despite possessing both putative NLSs. This was presumably due to a conformational change in the structure of the protein after deletion of the first 33 aa or may indicate the presence of a nuclear retention signal in this section of the protein.
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To assess further the efficacy of the KRRR motif as an NLS for other proteins, we constructed plasmids containing sequences of different lengths from the C terminus of pVI, as well as the full-length pVI, between two fluorescent proteins, EYFP and ECFP, or between EYFP and the puromycin resistance-encoding gene (pac) (Table 2). The EYFP and EYFPECFP plasmids were constructed from plasmid pdf20 and used for transient transfections of HeLa cells by using FuGene. The localization of the plasmid constructs 20 (control) and 20.120.6 was followed by using a DeltaVision restoration microscope. Successful nuclear accumulation was evident for all EYFP and EYFPECFP constructs with the exception of 20.2 (Fig. 5
). Construct 20.2, containing 34 aa from the C terminus of pVI, was found throughout the cytoplasm and nucleus. The most likely explanation for this, especially as construct 20.1 accumulated in the nucleus, is that the signal sequence is buried in between the two fluorescent proteins and is thus inaccessible. Thus, whilst the Pac protein does not hinder recognition of the NLS in the case of construct 20.1, ECFP, presumably due to its size, does. This is in accord with reports that the karyophilic domain needs to be exposed on the surface of the protein to function as an NLS (Roberts et al., 1987
).
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Fluorescent constructs enable the in vivo detection of adenain activity during infection
As well as containing the putative NLS, constructs 20.2 and 20.4 also contained an adenain cleavage site, whilst construct 20.6 contained two such sites. As these cleavage sites lay between two proteins with different fluorescent properties, these constructs had the potential to act as substrates that could be used to localize protease activity during infection. To determine whether the fusions could be cleaved by adenain, a rabbit reticulocyte lysate transcriptiontranslation system (Promega) was primed with constructs 20.120.6 and the expressed protein was incubated with purified recombinant adenain and pVI-CT. The different constructs were cleaved with varying efficiency: 20.1 and 20.2 were cleaved poorly, whereas 20.320.6 were cleaved more efficiently (results not shown). This was consistent with the observation above that the fusion protein containing the shortest (34 aa) insert from pVI was not localized to the nucleus as efficiently as those with a longer insert, possibly because steric factors hindered access to the relevant sequence motifs.
To test the possibility that the constructs with the larger inserts from pVI could act as markers of adenain activity, HeLa cells were transfected with construct 20.4 and 15 h later infected with Ad2. Fig. 6 shows the pattern of fluorescence 32 h after Ad2 infection. As expected, the intact fusion protein accumulated in the nucleus (Fig. 6a
) with the punctate distribution observed previously (Fig. 5
). Under the conditions used, the intact fusion proteins displayed a yellow fluorescence (Fig. 5f
) and the consequence of protease activity was evident with the appearance of red fluorescence from the separated EYFP fusion and green fluorescence from the pVI-CTECFP fusion (Fig. 6b and c
). These experiments with fluorescent substrates showed that adenain is proteolytically active in the nucleus approximately 3032 h after initial infection and that our synthetic fluorescent substrates could be used to assess in vivo protease activity at different points during adenoviral infection.
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DISCUSSION |
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The finding that the KRRR motif was not a major factor in the interaction of pVI-CT and adenain, nor in the development of catalytic activity by the complex, led us to investigate the possibility that it might act as an NLS. The results presented indicate that this sequence is capable of directing proteins whose distribution is normally cytoplasmic to the nucleus, and studies with PK defined the minimum requirement for nuclear accumulation to be the tripeptide KRR. Interestingly, the pattern of localization within the nucleus (a distinctive punctate appearance) was identical to that observed for pVI during adenovirus infection (Table 2), suggesting that the C-terminal region may have further specificity for targeting within the nucleus.
The viral protein pVI, from which pVI-CT was derived, contains two potential NLSs. That found in pVI-CT is close to the C terminus of the 250 aa protein and the other (KRPRP) is close to the centre at aa 131135. Although the KRRR sequence was sufficient for the nuclear targeting of PK (Fig. 4), EGFP and (provided they were part of a sufficiently extended linker) combinations of EYFP, ECFP and Pac (Fig. 5
) required both the KRPRP and KRRR signals for the nuclear localization of pVI. The inability of iVI(1239)EGFP to accumulate in the nucleus in a similar manner to pVIEGFP supported the general concept that the protease does not cleave pVI-CT until the maturation stage, which takes place following virion assembly in the nucleus.
Wodrich et al. (2003) provided evidence that the presence of two NESs, in addition to the two NLSs, allows pVI to shuttle between the nucleus and the cytoplasm, transporting the hexon protein to the nucleus in the process. Our data are in agreement in so far as they suggest the concept of pVI-CT as a nuclear targeting sequence, and these findings provide a rationale for the requirement for both NLSs in the nuclear targeting of pVI. This would suggest that if the ratio of NES to NLS is altered from 2 : 2 to 2 : 1 (as seen with the CMEGFP and MMEGFP constructs), the cytoplasmic accumulation of the fusion proteins is promoted. However, the pVI-CT constructs 20.1, 20.3 and 20.4 carried one NLS and one NES, and the finding of nuclear accumulation suggested the predominance of the NLS. In contrast to the results reported by Wodrich et al. (2003)
, we found no evidence of pVI leaving the nucleus following transfection (by using various fluorescent constructs) or following infection of cells with adenovirus (determined by immunofluorescence). In our studies, the distribution of the expressed pVI was consistent in both the transfections and infections.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 18 April 2004;
accepted 6 July 2004.
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