Adenovirus core protein VII contains distinct sequences that mediate targeting to the nucleus and nucleolus, and colocalization with human chromosomes

Tim W. R. Lee1, G. Eric Blair1 and David A. Matthews2

1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Department of Pathology and Microbiology, School of Medicine, University of Bristol, Bristol BS8 1TD, UK

Correspondence
Tim Lee
timlee{at}doctors.org.uk


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During adenovirus infection, following capsid dissociation, core protein VII enters the host cell nucleus complexed with adenovirus DNA. In order to determine whether protein VII may have an active role in this nuclear import, regions of the preVII gene were amplified by PCR, and further oligonucleotide mutants were designed with site-directed mutation of codons for the basic amino acids arginine and lysine. Fragments were cloned into a mammalian expression plasmid to express the peptides as N-terminal fusions to enhanced green fluorescent protein. Results demonstrate that preVII protein contains both nuclear and nucleolar targeting sequences. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, the data suggest that protein VII may bind to human chromosomes by means of two distinct domains, one sharing homology with the N-terminal regulatory tail of histone H3.


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Enclosed by approximately 12 capsid proteins the human adenovirus genome, comprising approximately 36 kb of linear double-stranded DNA, is non-covalently bound to the viral core proteins Mu, V, and VII (Vayda et al., 1983). Proteins VII and Mu are tightly associated with the viral DNA (Vayda et al., 1983; Chatterjee et al., 1985), whilst protein V may form a link between the viral DNA–core protein complex and the viral capsid (Matthews & Russell, 1998).

In human adenoviruses, both protein VII and Mu are encoded by the late transcription unit L2 (Alestrom et al., 1984). Mature protein VII of human adenovirus 2 and 5 contains 174 amino acids and is formed from its precursor by adenovirus-encoded protease-mediated cleavage of a 24 amino acid N-terminal segment (Sung et al., 1983; Alestrom et al., 1984; Webster et al., 1989). The mature protein has four highly basic domains containing arginine- and lysine-rich sequences, separated by predicted alpha-helices. The interaction of protein VII with DNA appears to be charge-based between these basic regions and the phosphate backbone of 90–150 DNA bp (Vayda & Flint, 1987), resulting in the DNA being considerably condensed as a result of superfolding (Black & Center, 1979; Sato & Hosokawa, 1984). Protein VII associates more efficiently with double-stranded DNA than single-stranded (Sato & Hosokawa, 1984). Binding of both proteins VII and Mu to DNA is not DNA sequence-specific (Russell & Precious, 1982).

During adenovirus infection, following capsid dissociation, protein VII remains complexed to adenovirus DNA, and this complex enters the nucleus via the nuclear pore (Greber et al., 1997). Protein VII inhibits adenovirus DNA synthesis (Korn & Horwitz, 1986) and transcription in vitro (Nakanishi et al., 1986) and thus is then likely to dissociate from DNA to allow these processes to occur. Antibodies raised against protein Mu for use in Western blots and ELISA cross-react with protein VII, presumably due to the homologous unusual arginine-rich sequences present in both proteins (Lunt et al., 1988). Protein VII has homologues in every adenovirus studied so far isolated either from mammals and birds, or from lower vertebrates (Zhang et al., 1991; Tarassishin et al., 1999; Farkas et al., 2002; Davison et al., 2003), and has significant functional and sequence similarity with histone H3 (Cai & Weber, 1993). Whether protein VII has any role beyond that of a DNA-binding protein remains unclear, but protein VII enhances the uptake and expression of naked DNA fourfold when used as a transfection adjuvant for mammalian cells (Wienhues et al., 1987), suggesting that it may be actively involved in the transport of DNA to the nucleus.

Nuclear and nucleolar targeting sequences have been examined in protein V (Matthews, 2001) and, since protein VII has similar arginine-rich regions, analysis of these putative targeting sequences was performed. In this study, we have determined that the amino acid sequence of protein VII and its precursor contains multiple nuclear and nucleolar localization signals.

In order to determine the intracellular targeting properties of regions of preVII protein, regions of the preVII gene were amplified from HAdV-2 DNA using oligonucleotide primers (primers on request) and a PCR kit (PFX; Gibco BRL). The resulting fragments, encoding a series of deletion mutants of the preVII protein, were cloned into a mammalian expression plasmid (pcJMA2egfp, kindly donated by J. Askham; Askham et al., 2000) to express the amino acid sequences produced as N-terminal fusions to enhanced green fluorescent protein (EGFP). We also evaluated several different monoclonal antibodies raised against protein VII but found none that were suitable for immunofluorescence-based localization studies (unpublished observations).

The plasmid constructs were transfected into HeLa cells grown at 37 °C with 5 % CO2 on glass coverslips in six-well dishes using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % foetal calf serum, penicillin (100 IU ml-1) and streptomycin (100 µg ml-1). The cells were transfected with 0·5 µg of each plasmid using Lipofectamine (Gibco BRL); 20 to 24 h after transfection the cells were fixed using 4 % (v/v in PBS) formaldehyde. Cells were washed in PBS, prior to the coverslips being mounted on Vectashield with 4',6-diamidino-2-phenylindole (DAPI; Vector Laboratories). EGFP-tagged proteins were detected with a Leitz Dialux microscope equipped with epifluorescence optics using a 63x/1·4 oil immersion lens and a Wild automatic camera system.

The deletion mutants of preVII generated, and the intracellular localization of the EGFP fusion products in HeLa cells, are shown in Fig. 1(A, C). For each fusion protein the indicated intracellular localization pattern was observed in almost all transfected cells. For comparison, untagged EGFP demonstrated a generalized diffuse distribution throughout the cell. Full-length preVII fused to EGFP (1–198EGFP) targeted the nucleolus, whereas mature protein VII (25–198EGFP) demonstrated nuclear targeting and was excluded from the nucleolus. As the precursor fragment 1–24EGFP does not exhibit any inherent nucleolar targeting properties, this inability of the mature VII fusion protein to target the nucleolus may be as a result of the evident nucleolar targeting signals within the protein being masked by differences in the tertiary structure of mature VII compared to preVII. The marked contrast between the intracellular localization of preVII and mature protein VII may suggest that these proteins perform different roles during infection. Alternatively, it is possible that the precursor fragment influences the folding of preVII in a manner that is key to the correct final conformation of mature VII after cleavage.



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Fig. 1. Identification of regions of preVII protein involved in nuclear and nucleolar targeting, and colocalization with human chromosomes in mitotic cells. (A) Schematic demonstrating the regions of protein VII fused to EGFP. On the left, each deletion mutant is identified by the amino acids from the full-length preVII sequence expressed N-terminal to the EGFP sequence. To the right, the intracellular distribution of each fusion protein in interphase cells is represented as No. (nucleolar), Np. (nucleoplasmic) or Cyt. (cytoplasmic), using an arbitrary scale, with EGFP alone for comparison. The intracellular localization of each fusion protein in mitotic cells relative to human chromosomes (Chromo.) is represented as either ‘colocalized’, or ‘excluded’ from area containing DAPI-stained cellular chromosomes. (B) Arginine- and/or lysine-rich sequences within protein VII that correspond to deletion mutants of protein VII that are able to independently direct EGFP to the nucleus and/or nucleolus. (C) Intracellular localization of four representative fusion proteins, compared with pattern observed in cells transfected with EGFP alone, demonstrating the pattern of fluorescence observed in these experiments. Panels (i)–(v) show DAPI staining; (vi)–(x) show EGFP expression. Panels (i), (vi) EGFP alone; (ii), (vii) preVII–EGFP; (iii), (viii) mature VII–EGFP; (iv), (ix) 25–54EGFP; (v), (x) 93–112EGFP. All images were scanned from colour print film and labelled using Adobe Photoshop 4.0.

 
Fusion protein 25–54EGFP demonstrated nuclear targeting (Fig. 1C), and had a nuclear localization sequence (NLS) type sequence within it (Fig. 1B). 93–112EGFP and 113–157EGFP displayed nucleolar targeting properties – both these regions of the protein are arginine-rich, but particularly 93–112 (Fig. 1B). As adenovirus is known to affect the structure and function of nucleoli (Castiglia & Flint, 1983; Walton et al., 1989; Matthews, 2001), this nucleolar targeting region was studied further. At low expression levels, targeting appeared nucleolar, with comparable nuclear and cytoplasmic background distribution. With increased expression, the cytoplasmic background staining became greater than that seen in the nucleoplasm. The contribution of the basic amino acids to the nucleolar targeting of 93–112EGFP was assessed by designing oligonucleotides encoding mutants of 93–112EGFP such that selected arginine or lysine codons were replaced by alanine codons (Fig. 2). Replacement of up to five arginines by alanines did not noticeably alter the intensity of nucleolar targeting; neither did rearranging the sequence such that the basic amino acids were equally distributed throughout the mutant peptide (with the overall amino acid content remaining unchanged). This latter observation would suggest that it is the charge to mass ratio that is responsible for the nucleolar targeting of these peptides, and not any sequence-specific interaction. All mutant clones were found to have comparable levels of protein expression on Western blot using antibody against EGFP (data not shown).



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Fig. 2. Mutational analysis of the nucleolar localization signal (NoLS) 93–112EGFP. The wild-type sequence is shown, with arginines and lysines highlighted in bold, followed by a series of mutants (numbered m1–m9) whereby selected arginine and/or lysine codons were replaced by alanine codons (highlighted in bold). Two further mutants were designed, one with all the arginine codons replaced by lysine codons (m10), and one with the same amino acid content as the wild-type, but with the codons for the basic amino acids equally distributed throughout the mutant peptide (m11). On the right, the intracellular distribution of each mutant fusion protein is represented as No. (nucleolar), Np. (nucleoplasmic) and/or Cyt. (cytoplasmic), using an arbitrary scale, with wild-type for comparison.

 
During these investigations it was noted that the protein VII–EGFP fusions colocalized with chromosomes in mitotic cells. To increase the proportion of cells visualized in mitosis, cell-cycle arrest was achieved in metaphase using colcemid (Urbani et al., 1995). Forty hours after transfection of plasmid constructs (as above), the HeLa cells were incubated for 8 h in colcemid (0·05 µg ml-1 in DMEM supplemented with 10 % foetal calf serum and antibiotics as above), prior to fixing and mounting as above. This protocol resulted in 10–20 % of DAPI-stained HeLa cells being in mitosis.

The intracellular localization of preVII–EGFP deletion mutants generated in mitotic HeLa cells relative to cellular chromosomes is shown in Figs 1(A) and 3(A). For comparison, untagged EGFP was excluded from areas containing DAPI-stained cellular chromosomes. Full-length preVII (1–198EGFP) colocalized with DAPI-stained cellular chromosomes, as did mature VII (25–198EGFP). Two regions within protein VII, 25–54 and 134–198, appeared to be responsible for this colocalization, as both these sequences fused to either EGFP or a Myc tag (data not shown) independently colocalized with cellular chromosomes (Fig. 3A, data shown for 25–54EGFP). The sequence 1–54EGFP was excluded from the chromosome-rich area, suggesting that the precursor fragment 1–24 masks the sequence 25–54 in this fusion protein. Other regions of preVII–EGFP that were excluded from the chromosome-rich areas within mitotic cells included 1V92–GFP, 55V92–GFP, 113V168–GFP, 134V168–GFP, and the highly basic nucleolar targeting sequence 93V112–GFP. Thus sequences within protein VII that colocalize with chromosomes in mitotic cells target the nucleus, rather than the nucleolus, in interphase cells (Fig. 3C). However, one fusion protein, 134V183–GFP, did not colocalize with chromosomes despite demonstrating nucleoplasmic localization in interphase cells.



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Fig. 3. Localization of VII 25–54-EGFP fusion protein in mitotic HeLa cells relative to cellular chromosomes, and possible functional implications. (A) Representative images demonstrating colocalization with cellular chromosomes (25–54EGFP), and exclusion from cellular chromosome-rich areas (EGFP alone). (B) Amino acid sequence of the region of preVII–EGFP from amino acids 25 to 54 that appears to colocalize with cellular chromosomes in mitotic cells. Sequence alignment of this region of HAdV-2 protein VII with other adenovirus serotypes to demonstrate high degree of similarity between serotypes (conserved residues highlighted in bold), and sequence alignment of key basic residues with H3 histones (indicated by *). Accession numbers: HAdV-2 (P03266), HAdV-5 (FOADH5), HAdV-11 (AAN62511), HAdV-40 (Q89532), canine AdV-1 (B46116), murine AdV-1 (O10440). (C) Suggested domain map of preVII. Sequences that were found to colocalize with human chromosomes demonstrated nuclear targeting properties. Nucleolar targeting sequences did not colocalize with human chromosomes.

 
Human chromatin consists of DNA complexed with histone proteins (Horn & Peterson, 2002). Whilst adenovirus core protein VII has been proposed to fulfil a histone-like role in condensing adenovirus DNA (Sung et al., 1983), this is the first report of protein VII colocalizing with human chromosomes. The possible significance of this colocalization remains to be explored; however, it is notable that other than the precursor fragment 1–24, the sequence 25–54 which colocalizes with chromosomes is the most highly conserved sequence between human, canine and mouse adenoviruses (Tarassishin et al., 1999), and shares some similarity with the N-terminal regulatory tail of histone H3 (Fig. 3B), a region responsible for transcriptional control (Cai & Weber, 1993; Strahl & Allis, 2000). Although non-mammalian adenoviruses such as duck and snake also demonstrate conservation in this region, similarity with histone H3 is less apparent (Farkas et al., 2002).

Previous studies have explored the binding of protein VII to adenovirus DNA using UV light cross-linking (Chatterjee et al., 1986a, b, c). These studies suggested that there were two DNA-binding domains within protein VII, one within the N-terminal half of the protein, and one within the C-terminal portion. Chatterjee et al. (1986b) showed that a fragment corresponding to the region 20–81 bound to DNA, and based on predicted structure, suggested that the N-terminal DNA-binding domain was in the region 55–67. Data presented in this study now suggest that this domain might correspond to the region 25–54 (Fig. 3C). The C-terminal domain was thought to be more predominant in the binding of the protein to adenovirus DNA within the virus core, and it was suggested that the three predicted alpha-helices, 114–124, 134–146 and 157–168, might be important as they are reminiscent of other DNA-binding domains such as Cro proteins and the E. coli lac repressor (Chatterjee et al., 1986a). The data presented in this study would be consistent with both 134–146 and 157–168 being necessary, but not sufficient, for DNA binding to occur, as 113–198EGFP and 134–198EGFP colocalized with cellular chromosomes in mitotic cells, whereas 134–183EGFP and 158–198EGFP did not colocalize. Taken together with this previous data concerning DNA binding, this study suggests that protein VII may bind directly to human DNA or to proteins associated with human DNA, at least during mitosis. Entry of quiescent cells into the S-phase of the cell cycle is an early event during adenovirus infection, when levels of protein VII are low. It is thus likely that high levels of protein VII would not normally be present in mitotic host cells, but the findings described in this paper may suggest an interaction during other phases of the cell cycle when chromatin is less organized.

In conclusion, human adenovirus core protein VII contains nuclear and nucleolar targeting signals. Such signals may be important in the delivery of adenovirus DNA to the host cell nucleus during adenovirus infection. Furthermore, data presented in this study suggest that protein VII may bind to human DNA in mitotic cells by means of two distinct domains.


   ACKNOWLEDGEMENTS
 
The research is supported by the Medical Research Council through personal Fellowships to D. A. M. and T. W. R. L.


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Received 1 August 2003; accepted 5 September 2003.