1 Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University, Taipei, Taiwan, Republic of China
2 Department of Microbiology and Immunology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China
Correspondence
Shiau-Ting Hu
tingnahu{at}ym.edu.tw
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ABSTRACT |
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INTRODUCTION |
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The dengue virion is approximately 50 nm in diameter and is composed of a single-stranded, positive-sense RNA genome and three structural proteins: the core (capsid, C), membrane (M) and envelope (E) proteins. The dengue viral genome is approximately 11 000 nt in length and encodes a large polypeptide, which is processed to form three structural and seven non-structural (NS) proteins. The order of genes encoding these proteins is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Chambers et al., 1990; Westaway et al., 1985
). The core protein has a molecular mass of 16 kDa and is the building block of the dengue nucleocapsid (Wang et al., 2002
).
Although the core proteins of various flaviviruses share very little sequence homology, they all are rich in basic amino acid residues (approx. 25 % Lys and Arg), which may be responsible for binding the core protein to the RNA genome (Chambers et al., 1990; Rice et al., 1985
). Such interaction has been demonstrated in Kunjin virus and hepatitis C virus (HCV). The core proteins of these two viruses of the Flaviviridae family have been shown to bind both 5'- and 3'-untranslated regions of their respective RNA genomes in vitro (Fan et al., 1999
; Khromykh & Westaway, 1996
; Shimoike et al., 1999
; Tanaka et al., 2000
). The RNA-binding ability of the dengue virus core protein has not been demonstrated.
The core protein of dengue virus type 2 (DEN2) has exactly 100 aa and has recently been shown by far-UV circular dichroism and NMR analyses to be a helical protein containing four -helices located at aa 2631, 4555, 6369 and 7496, respectively (Jones et al., 2003
). By analytical ultracentrifugation, NMR relaxation measurement and cross-linking with the lysine-specific reagent disuccinimidyl suberate, the DEN2 core protein has been shown to form homodimers in solution without the involvement of other viral components (Jones et al., 2003
). This result suggested that the DEN2 core protein has a domain responsible for the homotypic interaction similar to that of HCV (Matsumoto et al., 1996
; Nolandt et al., 1997
; Yan et al., 1998
) and tick-borne encephalitis (TBE) virus (Kofler et al., 2002
). In this study, we have identified the homotypic interaction domain of the DEN2 core protein and found that this domain is located at aa 3772, which overlaps the internal hydrophobic region (aa 4460) of the DEN2 core protein.
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METHODS |
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Co-immunoprecipitation.
To demonstrate homotypic interaction of the core protein, both FLAG-tagged and delta-tagged core proteins were expressed in the same cells. If the core proteins bind to each other, immunoprecipitation of the FLAG-tagged core protein will also precipitate the delta-tagged core protein and vice versa. Eight micrograms (in 8 µl water) each of the plasmid encoding a delta-tagged core gene (e.g. pDC1-100) and another plasmid encoding a FLAG-tagged core gene (e.g. pFC1-100) were mixed and incubated with 30 µl lipofectamine 2000 (Invitrogen Life Technologies) at room temperature for 20 min. This liposome/DNA mixture was added slowly to the medium of a HeLa cell culture in a 100 mm Petri dish. After 24 h incubation, the transfected cells were scraped into 300 µl binding buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris/HCl, pH 7·5) and lysed by repeated freezing and thawing. The cell debris was removed by centrifugation at 15 000 g for 10 min. Twenty microlitres Protein Aagarose (Sigma-Aldrich) was added to the clarified supernatant and the mixture was incubated for 3 h at 4 °C to remove proteins that may bind to Protein Aagarose non-specifically. After centrifugation at 1000 g for 1 min, the entire clarified supernatant was incubated with 10 µl (1 µg µl1) ANTI-FLAG M2 mAb (Sigma-Aldrich) or 10 µl (1 µg µl1) anti-delta HP6A1 mAb immobilized on Protein Aagarose for 5 h at 4 °C. The immune complexes thus formed were pelleted by centrifugation at 500 g for 5 min. The Protein Aagarose immune complex pellet was washed in 1 ml NET buffer (0·1 % Tween-20, 150 mM NaCl, 1 mM EDTA, 50 mM Tris/HCl, pH 7·5) to remove non-specifically bound proteins. The washed Protein Aagarose immune complexes were boiled in 20 µl sample buffer (Laemmli, 1970) and electrophoresed on a 13·5 % SDS-polyacrylamide gel followed by Western blotting using an HRP-conjugated secondary antibody and enhanced chemiluminescence reagent (Renaissance ECL kit; NEN Life Science Products) as the substrate, as described previously (Wang et al., 2002
). The Western blot was then exposed to an X-ray film to reveal bands that had reacted with the antibodies.
Nucleus translocation assay.
The nucleus translocation assay was performed as described previously (Wang et al., 2002). Plasmids encoding a FLAG-tagged and a delta-tagged core protein were co-transfected into HeLa cells as described above. The transfected cells were washed twice with cold PBS at 20 h post-transfection and fixed with methanol : acetone (1 : 1) mixture at 20 °C for 5 min. Nuclear localization of the core protein was determined by an immunofluorescence assay using either the ANTI-FLAG M2 mAb or the anti-delta epitope HP6A1 mAb as the primary antibody and FITC-conjugated anti-mouse antibody as the secondary antibody. The cells on slides were examined by confocal microscopy to determine the subcellular localization of the core protein.
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RESULTS |
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The same co-immunoprecipitation experiment was repeated using anti-delta mAb HP6A1, which was conjugated to Protein Aagarose to precipitate the core proteins. As described above, the immune complexes were electrophoresed and the Western blot of the gel was reacted with mAb M2 or mAb HP6A1, followed by a peroxidase-conjugated anti-mouse IgG (Fig. 2, lanes 79 and 1618). The results showed that anti-delta mAb (HP6A1)-precipitated core protein also reacted with ANTI-FLAG (mAb M2) antibody (Fig. 2
, lane 8), indicating that the FLAG-tagged core protein was co-precipitated with the delta-tagged core protein by the anti-delta antibody.
Localization of the homotypic interaction region of the DEN2 core protein by co-immunoprecipitation
To localize the homotypic interaction domain of the core protein, we constructed several recombinant plasmids (pFC1-84, pFC1-72, pFC15-100, pFC37-100 and pFC47-100) to express N- or C-terminal-truncated core proteins. pFC1-84 and pFC1-72 encoded FLAG-tagged C-terminal-truncated core proteins lacking aa 85100 and 73100, respectively. These two constructs were used to investigate the role of aa 85100 and 73100 containing helix IV in core-to-core interactions. The FLAG-tagged core proteins encoded by pFC15-100, pFC37-100 and pFC47-100 were missing aa 114, 136 and 146, respectively, and were used to determine whether these regions are involved in homotypic interactions of the core protein. Each of these plasmids was co-transfected with pDC1-100 into HeLa cells. The cell lysate of the transfected cells was then assayed for production of the FLAG-tagged truncated core protein and the ability of this core protein to bind the full-length delta-tagged core protein (the bait).
The Western blot of the cell lysate reacted with the anti-delta mAb HP6A1 revealed that the delta-tagged core protein DC1-100 was successfully expressed in the transfected cells (Fig. 3a, lanes 1723). Immunoprecipitation of the cell lysates was then performed using the Protein Aagarose-conjugated ANTI-FLAG mAb M2 to precipitate the FLAG-tagged core protein and the delta-tagged core protein DC1-100 that bound to it. The immune complexes were electrophoresed, blotted and reacted with ANTI-FLAG mAb M2 or anti-delta mAb HP6A1 as described above. The Western blot reacted with ANTI-FLAG mAb M2 revealed that all transfected cells expressed the FLAG-tagged truncated core proteins of expected sizes (Fig. 3a
, lanes 914). No core protein band that reacted with ANTI-FLAG antibody was seen in cells transfected with the control plasmid pFLAG-CMV-2 and pDC1-100 (Fig. 3a
, lane 15), indicating that the bands seen in Fig. 3
(lanes 914) were indeed core proteins. When the same Western blot was reacted with anti-delta antibody, various amounts of the delta-tagged core protein DC1-100 were detected in different samples (Fig. 3a
, lanes 16). Cells co-transfected with pFC15-100 and pDC1-100 were found to have the same amounts of delta-tagged core protein DC1-100 as those transfected with pFC1-100 and pDC1-100, indicating that aa 114 of the core protein were not essential for core-to-core homotypic interaction (Fig. 3a
, lanes 1 and 4). Cells co-transfected with pDC1-100 and pFC1-84, pFC1-72 or pFC37-100 were found to have greatly reduced amounts of protein DC1-100 that bound to these FLAG-tagged truncated core protein (Fig. 3a
, lanes 2, 3 and 5), suggesting that aa 85100, 73100 and 1536 play some roles in the homotypic interaction. Cells co-transfected with pDC1-100 and pFC47-100 only had trace amounts of delta-tagged core protein DC1-100 that bound to the FLAG-tagged truncated core protein FC47-100 (Fig. 3a
, lane 6). These results suggested that the domain most critical for homotypic interaction of the core protein is located at aa 3772.
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The region spanning aa 3772 overlaps the hydrophobic domain of the DEN2 core protein located at aa 4460 (Jones et al., 2003). To determine whether this hydrophobic domain is involved in core-to-core homotypic interaction, pFC
44-60 was constructed to express a FLAG-tagged core protein FC
44-60 lacking aa 44 (leucine) to 60 (proline). Although FC
44-60 was successfully expressed in cells transfected with pFC
44-60 (Fig. 4
, lane 5), it was unable to bind the bait FC1-100 in the co-immunoprecipitation experiment (Fig. 4
, lane 2). This result indicated that this hydrophobic domain is indeed involved in core-to-core homotypic interaction.
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DISCUSSION |
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The homotypic interaction domain that we have identified encompasses -helices II (aa 4555) and III (aa 6369) of the DEN2 core protein (Jones et al., 2003
). Using NMR, Ma et al. (2004)
recently determined the three-dimensional structure of the DEN2 core protein and predicted that helices II (aa 4555) and IV (aa 7496) would form the majority of the dimer contact surface. Although we did not find helix IV to be critical for homotypic interaction of the DEN2 core protein, deletion of the region encoding helix IV did greatly decrease the homotypic interaction ability of the core protein, as shown by the reduced binding of FLAG-tagged truncated core protein FC1-72 or FC1-84 to the delta-tagged DC1-100 (Fig. 3a
, lanes 2 and 3). We also found that truncation of helix I (aa 2631) reduced core-to-core interactions (Fig. 3a
, lane 5, FC37-100). This result is consistent with the prediction that the side chains of Val-26 and Leu-29 in helix I, Phe-47 and Val-51 in helix II and Ile-65, Arg-68 and Trp-69 in helix III form a hydrophobic interior that stabilizes the core homodimers (Ma et al., 2004
).
The internal hydrophobic sequence (aa 4460) is also located in the homotypic interaction domain that we have identified. The function of the DEN2 internal hydrophobic region has not been determined. It has been shown in vitro that the internal hydrophobic region of the DEN4 core protein is associated with the endoplasmic reticulum (ER) membrane, which may be the site for morphogenesis of DEN4 virus (Markoff et al., 1997). Based on the predicted three-dimensional structure of the DEN2 core protein, this internal hydrophobic region would create a large contiguous apolar patch that may interact with the ER membrane (Ma et al., 2004
). It is possible that DEN2 core protein binds to the ER membrane through this hydrophobic domain, forming a base for capsid assembly. The capsid may then capture the viral genome to form the viral nucleocapsid. Interaction of the core protein of Kunjin virus with the viral genome has been shown to be mediated through the basic amino acid residues present in both N and C termini of the core protein (Khromykh & Westaway, 1996
). Both the N- and C-terminal regions of the DEN2 core protein are also rich in basic amino acid residues, which may have a similar function in RNA binding to that of Kunjin virus. The HCV core protein has been shown to undergo heterotypic interactions with the HCV E1 protein (Lo et al., 1996
), in addition to homotypic interaction with itself (Matsumoto et al., 1996
; Nolandt et al., 1997
; Yan et al., 1998
). Heterotypic interactions with other structural proteins may influence viral budding and fusion processes (Lopez et al., 1994
). Whether the DEN2 core protein also undergoes heterotypic interactions remains to be investigated.
The core protein of TBE virus was predicted to possess two leucine zipper-like domains, which may mediate homotypic interaction (Kofler et al., 2002). Although structural analysis revealed that aa 4558 of the DEN2 core protein had homologies with the leucine zipper-like domains of TBE, this stretch of DEN2 core protein appears to be too short to form a leucine zipper motif. According to the predicted three-dimensional structure of DEN2 core protein, this region may form homodimers with helix II in an anti-parallel orientation (Ma et al., 2004
). Interestingly, the highly basic C-terminal helix IV of the DEN2 core protein is also predicted to dimerize forming an anti-parallel coiled-coil structure. The lack of a typical leucine zipper structure on the DEN2 core protein suggests that DEN2 core-to-core homotypic interactions are not as strong as those of TBE. This speculation is supported by our finding that the DEN2 core-to-core interaction was easily disrupted by washing the complex under more stringent conditions, such as washing with a triple-detergent lysis buffer (150 mM NaCl, 0·1 % SDS, 0·5 % sodium deoxycholate, 1 % NP-40, 100 µg PMSF ml1, 50 mM Tris/HCl, pH 8·0) (data not shown). In a yeast two-hybrid assay, the DEN2 core-to-core interaction was also found to be weak (data not shown). The identification of the DEN2 homotypic interaction domain in this study will enable a more detailed study of the assembly of the DEN2 nucleocapsid.
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ACKNOWLEDGEMENTS |
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Received 27 February 2004;
accepted 30 April 2004.
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