Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4811, Australia1
Nuclear Signalling Laboratory, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, Australia2
Author for correspondence: Subhash G. Vasudevan. Fax +61 7 47251394. e-mail Subhash.Vasudevan{at}jcu.edu.au
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Abstract |
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Introduction |
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The 11 kb single-stranded dengue virus RNA genome is capped but not polyadenylylated and encodes a single polyprotein including three structural and at least seven non-structural (NS) proteins in the order CprMENS1NS2ANS2BNS3NS4ANS4BNS5. The maturation of the polyprotein is achieved by the viral NS2BNS3 proteinase complex as well as host signal peptidases (Chambers et al., 1990
). Replication of dengue virus occurs at the membrane-associated replicase complex (RC). RC has been characterized extensively in other flaviviruses (e.g. Kunjin and YF) and includes NS1, NS2A, NS3, NS4A and NS5 (Mackenzie et al., 1998
; Westaway et al., 1997a
).
The NS3 protein (69 kDa) has protease activity localized within 167 N-terminal residues (Li et al., 1999 ). The immediate C-terminal region of the NS3 protease domain contains conserved motifs found in nucleoside triphosphate (NTP)-binding proteins and the DEXH family of RNA helicases. It was demonstrated recently that amino acid residues 160187 of NS3 are essential for its NTPase activity (Li et al., 1999
).
The NS5 protein (104 kDa) is predicted to contain at least two distinct domains: the N-terminal region is predicted to be an S-adenosyl-methionine (SAM) transferase domain, on the basis of similarity to several groups of methyltransferases from a wide variety of species (Koonin, 1993 ), whilst the C-terminal domain of NS5, from residue 455 onwards, contains eight highly conserved sequence motifs that have been recognized in many RNA-dependent RNA polymerases (RdRps; POL domain) (Koonin, 1991
). RdRp activity has been demonstrated for Escherichia coli-expressed dengue virus 1 NS5 protein (Tan et al., 1996
).
In dengue virus-infected cells, three RNA species have been observed: (i) the RNase-sensitive single-stranded genomic RNA, (ii) the double-stranded RNA replicative form (RF) and (iii) the partially RNase-sensitive replicative intermediate (RI) (Cleaves et al., 1981 ). The polymerase activity of NS5 probably requires the helicase and the NTPase activity of NS3 to replicate the genome from RF at RC. Also, the capping of the newly synthesized genome may require the combination of NS3 and NS5 activities, since capping is dependent on 5'-RNA triphosphatase activity (previously detected for NS3 from West Nile virus) (Wengler & Wengler, 1993
) as well as the putative SAM transferase activity of NS5. This is concordant with the demonstrated cytoplasmic form of NS5 interacting with NS3 both in vivo as well as in vitro (Kapoor et al., 1995
). However, the detailed molecular nature and function of this interaction in virus RNA replication has not been determined.
Although all suggested functions of NS5 are generally thought to occur in the cytosol, a hyperphosphorylated form of NS5 has been located in the cell nucleus (Kapoor et al., 1995 ). The NS5 of YF virus can also be detected in the nucleus (Buckley et al., 1992
) and recombinant NS5/NS5A of members of all three genera of the Flaviviridae have been shown to be phosphorylated by serine/threonine kinases (Morozova et al., 1997
). Other flavivirus proteins detected in the nucleus include the C (capsid) protein of dengue virus 2 (Bulich & Aaskov, 1992
) and the C and NS4B proteins of Kunjin virus (Westaway et al., 1997b
). However, hyperphosphorylation and nuclear localization of NS5 have not been demonstrated for other species of the genus Flavivirus. Proteins larger than 45 kDa in size generally require specific targetting signals called nuclear localization sequences (NLSs) in order to enter the nucleus (Jans, 1995
; Jans et al., 1991
) by using an intricate nuclear import machinery that involves recognition by the cellular NLS receptor, the importin-
/importin-
heterodimer, and other cellular factors, including the guanine nucleotide-binding protein Ran (Jans et al., 1998
). NLSs generally have no specific consensus sequence, consisting of hydrophilic residues that can be located at any position within the polypeptide that carries them as long as the NLS is accessible in the overall protein. The NLS of simian virus 40 (SV40) large tumour antigen (T-ag) is a typical example that consists of a single cluster of positively charged amino acids (PKKKRKV) (Jans et al., 1998
). A second type of NLS, called a bipartite NLS, has been identified in various nuclear-targetted proteins such as the steroid hormone receptors for glucocorticoid and progesterone. These consist of two clusters of positively charged residues that are separated by a 1012 amino acid residue spacer (Jans et al., 1998
). Importin-
binds the NLS region of nuclear-targetted proteins and importin-
then binds importin-
to enhance the former interaction. The complex consisting of the import proteins and the nuclear-targetted protein is transported through the nuclear pore and released in the nucleoplasm in a RanGTP-dependent process (Jans et al., 1998
; Ribbeck et al., 1999
).
We have demonstrated previously both in vivo and in vitro that a 37 amino acid domain of NS5 (aa 369405) can function as an NLS capable of targetting -galactosidase to the nucleus. This interdomain linker region of NS5 is recognized by the importin-
/
complex and contains a functional protein kinase CK2 phosphorylation site (threonine-395) that appears to inhibit nuclear targetting (Forwood et al., 1999
). The functional significance of nuclear transport and phosphorylation of NS5 has not been established and needs to be addressed. On the basis of our previous study, it was hypothesized that the NS5 NLS may be masked by interaction with NS3 in the early stages of replication. This site may become exposed at a late stage, either through conformational changes in NS5 resulting from hyperphosphorylation at serine residues or through autoproteolysis of NS3 that may provide access to the NLS for importins.
The picture that is emerging is of the RC as a protein machine, where many critical proteinprotein contacts and also RNAprotein interactions may underpin its replication function. The fine molecular mapping of these interactions is important both as a means to characterize them to reveal their significance in the replication mechanism and also to investigate the suitability of these interaction sites as specific targets for new antiviral compounds. The yeast two-hybrid (Y2H) system detects proteinprotein interactions in vivo in yeast by taking advantage of the modular nature of the transcription factor GAL4 (Ma & Ptashne, 1987 ). Briefly, a protein of interest is fused to a DNA-binding domain (BD), while a second protein is fused to a transcription-activation domain (AD). If the two proteins interact, the chimeric complex couples the BD domain bound at an upstream sequence element to the AD domain, which activates transcription of a reporter gene(s), e.g. lacZ or HIS3 (Chien et al., 1991
). The Y2H system has also previously been shown to be suitable for mapping interactions between the polymerase and helicase of the positive-stranded brome mosaic virus (OReilly et al., 1997
).
In this paper, we report the characterization of the interaction between NS3 and NS5 from dengue virus type 2 strain TSV01 by mapping the binding sites between these proteins using the Y2H system. Furthermore, we investigate the Y2H interaction for NS5 with the host-encoded nuclear import proteins, demonstrating for the first time that NS5 interacts with importin-. This interaction is verified and shown to occur independently of importin-
in pull-down assays. The function of the observed interactions, specifically a competitive regulatory role, is discussed.
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Methods |
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Isolation of NS3 and NS5 cDNAs.
Aliquots of the dengue 2 virus TSV01 RNA template (5 µg) were annealed with 2 pmol of the reverse gene-specific primers for NS3 or NS5 (Table 1). cDNA synthesis was performed with SuperScript II RNase Hreverse transcriptase according to manufacturers instructions (Life Technologies). The NS3 and NS5 gene-specific primers (Table 1
) were used to amplify the NS3 and NS5 genes separately from the TSV01 cDNA, using the EXPAND high-fidelity PCR enzyme mixture (Boehringer Mannheim). The amplified genes were subsequently inserted into the pGEM-T vector (Promega) to produce plasmids pMJNS3 AND pGEM-TNS5. The nucleotide sequences of the inserts in pMJNS3 and pGEM-TNS5 were identical to the nucleotide sequence obtained directly from RTPCR products of TSV01. The plasmid pMJNS5 was generated from pGEM-TNS5, by digestion with the restriction endonuclease SacII in order to remove an 8 bp non-coding region between the SacII site in the NS5 forward primer (Table 1
) and the same site within the pGEM-T vector, followed by ligation to reclose the plasmid. The two plasmids pMJNS3 and pMJNS5 were used in the construction of the various Y2H plasmids as well as the expression plasmids for the production of HISNS3 and HISNS5 (see Fig. 1
) in this study.
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The Y2H assay for reporter gene activity.
Y2H interactions were assayed with the in vitro plate filter lift assay (Clontech). Interactions were assayed on at least five independent transformants in both of Saccharomyces cerevisiae strains Y190 and Y187 (Table 1) to give conclusive results, but the data obtained with strain Y190 are shown exclusively in the figures since the interaction was stronger in that strain. Yeast colonies were patched onto SD minimal media lacking leucine or tryptophan in order to select for plasmids originating from pACT2 or pAS2-1, respectively. The plates were incubated at 30 °C for 48 h and the cells were transferred to nitrocellulose membrane and assayed according to the Yeast Protocol Manual (Clontech) with the modification of three freezethaw cycles. The development of the blue colour resulting from
-galactosidase activity started within 1 h for strong interactions, but required up to 8 h for the weaker interactions. The negative controls did not develop any colour for at least 24 h.
Detection of proteins expressed in yeast.
Yeast cells transformed with plasmids encoding the hybrid proteins were grown overnight at 30 °C in a suitable SD selection liquid medium. The cultures were inoculated 1:10 into YPD medium and grown to an OD600 of 0·6. The cells were collected on ice, frozen in liquid nitrogen and stored at -80 °C until required for protein extraction. Proteins were prepared by the ureaSDS extraction method (Printen & Sprague, 1994 ), separated on 10% SDSPAGE (Laemmli, 1970
), transferred to nitrocellulose membrane and detected with monoclonal AD or BD antibodies (Clontech) visualized by enhanced chemiluminescence (ECL) (PharmaciaAmersham Biotech).
Glutathione S-transferase (GST) pull-down assays of NS5 with GST-tagged importin-
.
Histidine-tagged NS5 and NS3 (HISNS5, HISNS3) were expressed in E. coli strain AD494 (DE3) (RIL) (Table 1) and purified by nickel-chelating chromatography (M. Johansson, A. J. Brooks and S. G. Vasudevan, unpublished), whereas GSTimportin-
(GSTImp
) was expressed and purified as described previously (Chan et al., 1998
; Forwood et al., 1999
). GSTImp
or the control protein GST11 (Catmull et al., 1992
) at 2 µg/µl (final concentration) in buffer A (20 mM TrisHCl, pH 7·9, 140 mM NaCl, 20 mM MgCl2 and 0·1% Triton X-100) was supplemented with NS5 at 120 ng/µl (final concentration). GlutathioneSepharose 4B (PharmaciaAmersham Biotech) was added to the proteins, which were then incubated with gentle agitation at 4 °C for 14 h. The beads were washed five times with buffer A and proteins were eluted with 40 mM reduced glutathione in buffer A. The eluted fractions were analysed on 10% SDSPAGE, transferred to nitrocellulose membrane and detected with mouse polyclonal NS5 antibody (M. Johansson and S. G. Vasudevan, unpublished) visualized by ECL. The protein concentration used in the pull-down assays was dependent on the amount of pure NS5 available; however, the specificity of the interaction was confirmed by the competition studies (see below). Furthermore, many pull-down experiments reported in the literature (e.g. Chung et al., 2000
) have used in vitro transcriptiontranslation products that were also in the same range of protein concentration. Competition assays were set up essentially as described above, but HISNS3 (equimolar or 10-fold excess compared with NS5) or BSA (equimolar with NS5) was added with HISNS5 in the presence of GSTImp
and glutathioneSepharose 4B.
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Results |
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ADNS3(1303) (Fig. 1), which contains the protease domain as well as the protease-sensitive site mentioned above, was expressed in yeast (data not shown), but showed no detectable reporter activity when tested with the BDNS5 constructs, suggesting that the NS5 interaction site is indeed within the C-terminal region of NS3.
NS5 interacts with the nuclear import receptor importin-
We have demonstrated previously that the interdomain linker region (residues 369405) of NS5 functions as an NLS both in vivo and in vitro (Forwood et al., 1999 ) and is recognized by the importin-
/importin-
heterodimeric complex. A number of recent studies have shown that importin-
can interact directly with nuclear-targetted proteins independently of importin-
to mediate their nuclear import (Chan et al., 1998
; Henderson & Percipalle, 1997
; Tiganis et al., 1997
). We decided to use the Y2H assay to examine the interaction of NS5 with importins, with particular interest in mapping the potential sites of interaction in relation to the NS5NS3 interaction site.
Full-length importin- and an N-terminally truncated importin-
[Imp
(262876)], both fused to BD, were analysed against ADNS5. No reporter activity was detected using full-length BDimportin-
[BDImp
(1876)] with ADNS5 (Fig. 4a
). It has been shown previously that BDImp
(1876) and ADimportin-
(ADImp
) do not interact in the Y2H system (Fig. 4b
), due to the fact that the N-terminal domain of importin-
can bind RanGTP, which prevents the importin-
/importin-
interaction (Herold et al., 1998
). In contrast, BDImp
(262876), which lacks the ability to bind Ran, shows strong interaction with importin-
(Fig. 4c
). Interestingly, BDImp
(262876) also exhibited an interaction with ADNS5 (Fig. 4d
, e
), implying that nuclear import of NS5 might be mediated directly by importin-
. ADNS5(320900) showed similar reporter activity to the full-length NS5 (Fig. 4f
), whereas the truncation ADNS5(406900) showed no detectable activity (Fig. 4g
). To test whether the previously demonstrated NLS sequence, NS5(369405), may be the region responsible for interaction with importin-
, ADNS5(1368) and ADNS5(1405) were analysed, both of which were expressed at similar levels in yeast (data not shown). Unexpectedly, ADNS5(1368) (Fig. 4h
), which does not include the previously characterized NS5 NLS, showed reporter activity upon interaction with BDImp
(262876) that was much greater than that observed with ADNS5(1405) (Fig. 4i
) or ADNS5 (Fig. 4d
, e
). These data indicate that the region immediately N-terminal of the previously characterized NLS is critical for binding of importin-
to NS5. Human importin-
and -
3 were also analysed for their ability to interact with NS5 in the Y2H system, but neither showed reporter gene activity indicative of interaction with either ADNS5 or the truncated constructs of NS5 (data not shown).
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Mapping of the NS5 interaction regions by Y2H analysis
Analyses of the full-length and domain constructs of NS5 indicated that the region 320368/(405) is important for the NS5NS3 and NS5importin- interactions. Plasmids expressing the smaller constructs ADNS5(369405), ADNS5(320368) and ADNS5(320405) were constructed to determine more precisely the site of protein interactions within this region. ADNS5(369405) was expressed well (data not shown) but was found to interact only weakly with importin-
(Fig. 6a
). Although the reporter activity was low, it could be detected reproducibly, and a similar result was obtained for plasmids expressing the reciprocal peptides (data not shown). No reporter gene activity was detected for BDNS3(303618) interacting with ADNS5(369405) (Fig. 6d
) or the reciprocal peptides (data not shown). ADNS5(320368) was expressed at lower levels than ADNS5(369405) but did show interactions with both BDImp
(262876) and BDNS3(303618), indicating that the region including residues 320368 is important for interactions of NS3 as well as importin-
with NS5 (Fig. 6b
, e
). The longer peptide ADNS5(320405) was expressed poorly (data not shown) and showed no reporter activity with BDImp
(262876) (Fig. 6c
). On the other hand, some interaction could be detected with BDNS3(303618) (Fig. 6f
). The truncation constructs demonstrate quite clearly that both NS3 and importin-
interact with NS5 within
50 amino acids.
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Discussion |
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In terms of specific results, we made the interesting observation that, in contrast to truncated versions, full-length NS5 and full-length NS3 do not interact in the Y2H assay. This can probably be attributed to proteolytic cleavage of NS3 (around residue 150), liberating the NS3 C terminus from the GAL4 BD to compete with the full-length protein for NS5, which is consistent with the fact that NS3 (residues 303618) interacts with full-length NS5 as well as with various truncations of NS5. In fact, the results obtained from the reporter assays indicate that the interaction of the NS3 C-terminal domain is stronger in the presence than in the absence of the N-terminal domain of NS5, since the truncation construct ADNS5(406900) showed the weakest interaction with BDNS3(303618). The data clearly indicate that the region N-terminal of the NS5 NLS and POL domain region is responsible for NS3 binding. Similarly, Y2H studies indicate that interaction of brome mosaic virus helicase-like protein 1a and the 2a polymerase is mediated by a region N-terminal of the polymerase homology region (OReilly et al., 1997 ).
We found that an importin- construct with a deleted N terminus interacted strongly with NS5, although previous in vitro studies had indicated that the NS5 NLS is recognized with reasonably high affinity by the importin-
/
complex (Forwood et al., 1999
). Several recent reports indicate that there are a number of different pathways by which nuclear-targetted proteins can be transported to the nucleus in addition to the well-characterized importin-
/
heterodimer-mediated pathway (Jans et al., 1998
). The ability of importin-
to interact directly with NS5 independently of importin-
suggests that the latter may be transported to the nucleus by the importin-
-mediated pathway. This direct interaction of importin-
with NS5 independent of importin-
was confirmed in pull-down assays. Interestingly, it is possible to find some significant similarity within the NS5 region that interacts with importin-
to the minimal importin-
-binding regions of parathyroid hormone-related protein (PTHrP) and T-cell protein tyrosine phosphatase (TCPTP) (Lam et al., 1999
). This region of NS5 is aligned against a comparison of the PTHrP region with TCPTP as well as human immunodeficiency virus type 1 (HIV-1) Rev and GAL4 in Fig. 7
.
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A recent report showed that NS5A from HCV, a member of the family Flaviviridae, interacts with the importin- homologue karyopherin-
3 (44·4% similarity, 17·6% identity) (Chung et al., 2000
). This interaction has been characterized by using the Y2H system and shown to occur between the N-terminal region of NS5A and the C-terminal part of karyopherin-
3 (Chung et al., 2000
). HCV NS5A is not detected in the nuclei of infected cells, although a region resembling an NLS has been fused N-terminally to
-galactosidase and shown to target it to the nucleus (Ide et al., 1996
). A putative hijacking role for NS5A was suggested, whereby the viral protein functions by sequestering nuclear karyopherin-
3 (RanBP5/importin 5) so that it is not available for other cellular functions (Chung et al., 2000
). There is no apparent similarity between the interacting sequence of NS5A and the dengue virus NS5 sequence to suggest a similar role, not even within NS5 residues 320405.
There is no known role for nuclear localization of dengue virus NS5, but the facts that nuclear localization does occur in vivo in dengue virus-infected cells (Kapoor et al., 1995 ) and that the protein contains a functional NLS (Forwood et al., 1999
) clearly imply that it is significant. Detailed investigation using the approaches described here should help to unravel the mechanism and events that trigger the nuclear import of NS5 and may provide a new target for the development of antiviral agents. This process necessarily involves investigation of protein interaction sites that may be amenable to high through-put assays in order to find suitable inhibitors. The Y2H system clearly represents an attractive genetic screen to do exactly this, and hence will be highly useful in future, as soon as the functional consequences of the different competing NS3NS5importin interactions are fully understood through complementary studies involving reverse genetics with infectious clones.
In conclusion, the NS3 protein must work in concert with NS5 for replication of the viral genome, since the RF is a double-stranded RNA. This process occurs in the perinuclear membrane of infected cells. Since NS3 also interacts with the importin--binding site defined in this work, it is possible that an additional role for NS3 may be to ensure that NS5 remains in the cytoplasmic environment, where it is necessary for virus replication and capping. Detailed characterization of this region by specific mutagenesis as well as nuclear localization studies with recombinant NS5 are currently in progress in this laboratory.
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Acknowledgments |
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Received 4 September 2000;
accepted 20 December 2000.