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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ABSTRACT |
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MAIN TEXT |
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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 90150 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 (1198EGFP) targeted the nucleolus, whereas mature protein VII (25198EGFP) demonstrated nuclear targeting and was excluded from the nucleolus. As the precursor fragment 124EGFP 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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The intracellular localization of preVIIEGFP 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 (1198EGFP) colocalized with DAPI-stained cellular chromosomes, as did mature VII (25198EGFP). Two regions within protein VII, 2554 and 134198, 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. 3
A, data shown for 2554EGFP). The sequence 154EGFP was excluded from the chromosome-rich area, suggesting that the precursor fragment 124 masks the sequence 2554 in this fusion protein. Other regions of preVIIEGFP that were excluded from the chromosome-rich areas within mitotic cells included 1V92GFP, 55V92GFP, 113V168GFP, 134V168GFP, and the highly basic nucleolar targeting sequence 93V112GFP. 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, 134V183GFP, did not colocalize with chromosomes despite demonstrating nucleoplasmic localization in interphase cells.
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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 2081 bound to DNA, and based on predicted structure, suggested that the N-terminal DNA-binding domain was in the region 5567. Data presented in this study now suggest that this domain might correspond to the region 2554 (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, 114124, 134146 and 157168, 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 134146 and 157168 being necessary, but not sufficient, for DNA binding to occur, as 113198EGFP and 134198EGFP colocalized with cellular chromosomes in mitotic cells, whereas 134183EGFP and 158198EGFP 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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Askham, J. M., Moncur, P., Markham, A. F. & Morrison, E. E. (2000). Regulation and function of the interaction between the APC tumour suppressor protein and EB1. Oncogene 19, 19501958.[CrossRef][Medline]
Black, B. C. & Center, M. S. (1979). DNA-binding properties of the major core protein of adenovirus 2. Nucleic Acids Res 6, 23392353.[Abstract]
Cai, F. & Weber, J. M. (1993). Primary structure of the canine adenovirus PVII protein: functional implications. Virology 193, 986988.[Medline]
Castiglia, C. L. & Flint, S. J. (1983). Effects of adenovirus infection on rRNA synthesis and maturation in HeLa cells. Mol Cell Biol 3, 662671.[Medline]
Chatterjee, P. K., Vayda, M. E. & Flint, S. J. (1985). Interactions among the three adenovirus core proteins. J Virol 55, 379386.[Medline]
Chatterjee, P. K., Vayda, M. E. & Flint, S. J. (1986a). Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo. J Mol Biol 188, 2337.[Medline]
Chatterjee, P. K., Yang, U.-C. & Flint, S. J. (1986b). Comparison of the interactions of the adenovirus type 2 major core protein and its precursor with DNA. Nucleic Acids Res 14, 27212735.[Abstract]
Chatterjee, P. K., Vayda, M. E. & Flint, S. J. (1986c). Adenoviral protein VII packages intracellular viral DNA throughout the early phase of infection. EMBO J 5, 16331644.[Abstract]
Davison, A. J., Benkö, M. & Harrach, B. (2003). Genetic content and evolution of adenoviruses. J Gen Virol 84, 28952908.
Farkas, S. L., Benkö, M., Élö, P., Ursu, K., Dán, Á., Ahne, W. & Harrach, B. (2002). Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus. J Gen Virol 83, 24032410.
Greber, U. F., Suomalainen, M., Stidwell, R. P., Boucke, K., Ebersold, M. W. & Helenius, A. (1997). The role of the nuclear pore complex in adenovirus DNA entry. EMBO J 16, 59986007.
Horn, P. J. & Peterson, C. L. (2002). Chromatin higher order folding: wrapping up transcription. Science 297, 18241827.
Korn, R. & Horwitz, M. S. (1986). Adenovirus DNA synthesis in vitro is inhibited by the virus-coded major core protein. Virology 150, 342351.[Medline]
Lunt, R., Vayda, M. E., Young, M. & Flint, S. J. (1988). Isolation and characterization of monoclonal antibodies against the adenovirus core proteins. Virology 164, 275279.[Medline]
Matthews, D. A. (2001). Adenovirus protein V induces redistribution of nucleolin and B23 from nucleolus to cytoplasm. J Virol 75, 10311038.
Matthews, D. A. & Russell, W. C. (1998). Adenovirus core protein V is delivered by the invading virus to the nucleus of the infected cell and later in infection is associated with the nucleoli. J Gen Virol 79, 16711675.[Abstract]
Nakanishi, Y., Maeda, K., Ohtsuki, M., Hosokawa, K. & Natori, S. (1986). In vitro transcription of a chromatin-like complex of major core protein VII and DNA of adenovirus serotype 2. Biochem Biophys Res Commun 136, 8693.[Medline]
Russell, W. C. & Precious, B. (1982). Nucleic acid binding properties of adenovirus structural polypeptides. J Gen Virol 63, 6979.[Abstract]
Sato, K. & Hosokawa, K. (1984). Analysis of the interaction between the DNA and major core protein in adenovirus chromatin by circular dichroism and ultraviolet light induced cross-linking. J Biochem 95, 10311039.[Abstract]
Strahl, B. D. & Allis, C. D. (2000). The language of covalent histone modifications. Nature 403, 4145.[CrossRef][Medline]
Sung, M. T., Cao, T. M., Coleman, R. T. & Budelier, K. A. (1983). Gene and protein sequences of adenovirus protein VII, a hybrid basic chromosomal protein. Proc Natl Acad Sci U S A 80, 29022906.[Abstract]
Tarassishin, L., Szawlowski, P., McLay, J., Kidd, A. H. & Russell, W. C. (1999). Adenovirus core protein VII displays a linear epitope conserved in a range of human adenoviruses. J Gen Virol 80, 4750.[Abstract]
Urbani, L., Sherwood, S. W. & Schimke, R. T. (1995). Dissociation of nuclear and cytoplasmic cell cycle progression by drugs employed in cell synchronization. Exp Cell Res 219, 159168.[CrossRef][Medline]
Vayda, M. E. & Flint, S. J. (1987). Isolation and characterization of adenovirus core nucleoprotein subunits. J Virol 61, 33353339.[Medline]
Vayda, M. E., Rogers, A. E. & Flint, S. J. (1983). The structure of nucleoprotein cores released from adenovirions. Nucleic Acids Res 11, 441460.[Abstract]
Walton, T. H., Moen, P. T., Jr, Fox, E. & Bodnar, J. W. (1989). Interactions of minute virus of mice and adenovirus with host nucleoli. J Virol 63, 36513660.[Medline]
Webster, A., Russell, S., Talbot, P., Russell, W. C. & Kemp, G. D. (1989). Characterization of the adenovirus proteinase: substrate specificity. J Gen Virol 70, 32253234.[Abstract]
Wienhues, U., Hosokawa, K., Hoveler, A., Siegmann, B. & Doerfler, W. (1987). A novel method for transfection and expression of reconstituted DNAprotein complexes in eukaryotic cells. DNA 6, 8189.[Medline]
Zhang, C. L., Nagaraja, K. V., Sivanandan, V. & Newman, J. A. (1991). Identification and characterization of viral polypeptides from type-II avian adenoviruses. Am J Vet Res 52, 11371141.[Medline]
Received 1 August 2003;
accepted 5 September 2003.