Department of Microbiology1 and Department of Biochemistry and Molecular Biology2, Monash University, PO Box 53, Victoria 3800, Australia
Author for correspondence: Peter Wright. Fax +61 3 9905 4811. e-mail Peter.Wright{at}med.monash.edu.au
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Abstract |
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Introduction |
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The flavivirus genome is a positive-sense RNA molecule of approximately 11000 nucleotides encoding the proteins CprMENS1NS2ANS2BNS3NS4ANS4BNS5 in a single long open reading frame. Co- and post-translational polyprotein processing by host and viral proteinases generate three structural proteins, namely C (capsid), M (membrane) and E (envelope), and seven nonstructural (NS) proteins, namely NS1 through to NS5 (reviewed by Rice, 1996 ).
The focus of this paper is the viral NS2B/3 proteinase of Dengue virus type 2 (DEN-2). NS2B/3 cleaves at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions. It also cleaves within C, NS2A, NS4A and NS3, in the latter case producing NS3' and NS3' (Arias et al., 1993 ; Teo & Wright, 1997
). Proteolysis occurs following a pair of basic amino acids or GlnArg and preceding either Gly, Ser or Ala (Rice, 1996
). The motifs and catalytic triad typical of a trypsin-like serine proteinase are located in the N-terminal one-third of NS3 (Bazan & Fletterick, 1989
); the X-ray crystal structure for this part of NS3 was described recently (Murthy et al., 1999
). The active form of the viral proteinase is a complex between NS3 and NS2B (Preugschat et al., 1990
; Falgout et al., 1991
). A hydrophilic region of 40 amino acids in NS2B containing a short central hydrophobic segment is required for the association of NS2B with NS3 and for enzyme activity (Falgout et al., 1993
; Chambers et al., 1993
; Yusoff et al., 2000
). Similarly, an NS3-containing complex is an active proteinase of Hepatitis C virus (HCV), which also belongs to the family Flaviviridae, but to the genus Hepacivirus. In this case the complex is formed between NS3 and NS4A (Failla et al., 1995
; Lin et al., 1995
).
Initial studies on the cleavage of the flavivirus polyprotein targeted either the four regions of homology shared between serine proteinases and the flavivirus NS3 protein, or the cleavage sites in the polyprotein (Chambers et al., 1990 ; Valle & Falgout, 1998
). However, in this study, seven locations in NS3 outside these regions and sites were chosen for mutagenesis. By avoiding motifs containing the catalytic triad and residues known to be involved in substrate binding, it was reasoned that sites involved in NS2BNS3 interaction may be mutated and that suitable modification at such sites had the potential to reduce, without abolishing, proteinase activity and virus replication. Virus mutants of this type are candidates for incorporation into live vaccine strains of DEN-2 and other flaviviruses. Mutations were tested for their effects on NS2B/3 proteinase activity by transient expression of the NS2B/3 genes in COS cells, virus replication by the incorporation into genomic-length DEN-2 cDNA or both. Results are interpreted below with reference to the location of the mutations mapped on the X-ray crystal structure of the DEN-2 NS3 proteinase (Murthy et al., 1999
) and a model of the NS2B/3 complex.
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Methods |
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Transient expression of DEN-2 genes in COS cells.
The vector pSV.SPORT 1 (Gibco BRL) was used to express DEN-2 cDNA (strain New Guinea C) encoding NS2B/NS3. The construction of plasmid pSV.NS2B/3 (S2, the parental NS2B/NS3 construct) (Fig. 1) was described previously (Teo & Wright, 1997
). The nucleotide numbering used to describe the mutants below follows that of Irie et al. (1989)
. Four mutant constructs derived from pSV.NS2B/3 (S2) were prepared by replacing the EcoRV4331BstBI5070 fragment (Fig. 1
) with a mutated fragment prepared by overlap extension PCR (Ho et al., 1989
), i.e. constructs pSV.NS2B/31720 (S1720), pSV.NS2B/33236 (S3236), pSV.NS2B/36366 (S6366) and pSV.NS2B/39596 (S9596); the plasmid pSV.NS2B/3179181 (S179181) was prepared by conventional PCR using a mutagenic primer overlapping the BstBI5070 site. The PCR-derived regions of all clones were sequenced. The plasmid pSV.NS2B/33236 was initially designed to encode only the substitutions G32A and Y33A. However, an additional I36V change was introduced during PCR.
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Insertion of mutations into genomic-length DEN-2 cDNA.
The plasmid pDVWS501 containing genomic-length New Guinea C strain DEN-2 cDNA has been described in detail (Gualano et al., 1998 ). For these experiments, transient expression was used to examine the effects of five mutations in the NS3 proteinase on proteolytic activity. Three mutations were selected and then inserted into genomic-length DEN-2 cDNA to study their effects on virus replication. Two additional charged-to-alanine mutations that were external to proteinase motifs were also inserted into the genomic-length DEN-2 cDNA (Fig. 1
).
The plasmid pDVWS501NS33236 was prepared by replacing the NheI2544NsiI4700 fragment of pDVWS501 with a mutated fragment prepared by overlap extension PCR (Fig. 1). The other four mutations were initially constructed in the subclone pDVSO8298 prior to ligation into pDVWS501. This strategy was devised following consideration of the available restriction enzyme sites. Plasmid pDVSO8298 contained DEN-2 cDNA corresponding to nucleotides 4494 (upstream of NsiI4700) to 8744 (downstream of StuI7874) cloned into XbaI/KpnI-digested pSPORT 1 (Gibco BRL). cDNA encoding mutations KRIE (6366) (underlined residues changed to alanine) or EDD (179181) in the NS3 hydrophilic regions was cloned into pDVSO8298 by removing the mutated NsiI4700PpuMI5854 fragment from the corresponding pSV.NS2B/3 plasmid and ligating this into NsiI4700/PpuMI5854-digested pDVSO8298. The two remaining charged-to-alanine mutations, EGEE (9194) and EKSIE (169173), were introduced into NsiI4700/PpuMI5854-digested pDVSO8298 as overlap extension PCR fragments. All four mutants (NsiI4700StuI7874-mutated fragments) were then removed from the appropriate pDVSO8298 plasmid and ligated into NsiI4700/StuI7874-digested pDVWS501. PCR-derived regions were sequenced.
Production of virus from genomic-length cDNA.
Procedures for transcription of RNA, electroporation and immunofluorescence of BHK-21 cells and passaging of virus in C6/36 cells have been described previously (Gualano et al., 1998 ). Briefly, capped transcripts were produced from plasmids containing genomic-length DEN-2 cDNA using the Promega RiboMAX kit. Approximately 710 µg of transcript RNA and 50 µg of carrier tRNA were electroporated into BHK-21 cells, which were then incubated at 33 °C or 37 °C. Cells were examined for immunofluorescence 4 to 6 days later using anti-E monoclonal antibodies (Gruenberg & Wright, 1992
). After 7 days, the culture medium was used to infect C6/36 cells. The culture medium from these infected C6/36 cells was then used 4 to 5 days later to initiate a second passage. When approximately 50% of the cells exhibited cytopathic effects, or 5 days later if no cytopathic effects were visible, these second passage virus stocks were titred by plaque assay on C6/36 cells.
Each virus was derived at least twice from its parental construct. To confirm that each mutation was present after electroporation and passaging, total RNA was extracted from infected C6/36 cells or supernatant and RTPCR of viral RNA was performed (Gualano et al., 1998 ; Pryor et al., 1998
). The complete NS2B and NS3 genes were sequenced to confirm the presence of the introduced mutation and the absence of any other changes that may have been introduced during virus passaging.
Co-ordinates and calculations.
The crystal structures of the DEN-2 NS3 serine proteinase (protein database identifier 1BEF; Murthy et al., 1999 ) and the HCV NS3/NS4A proteinaseco-factor complex (protein database identifier 1NS3; Yan et al., 1998
) were obtained from the protein database (Bernstein et al., 1977
; Berman et al., 2000
).
The model between the DEN-2 NS3 proteinase and a portion of the NS2B co-factor was generated using the Quanta/CHARMm software (MSI). The peptide sequence G69SSPILSITISE80 within NS2B corresponds to the portion of the NS4A co-factor seen in the structure of the HCV proteinase (Brinkworth et al., 1999 ). The DEN-2 and HCV proteinases were superimposed and the NS4A peptide within the HCV proteinase structure was used as a template to model the NS2B sequence (GSSPILSITISE) into the DEN-2 NS3 proteinase. The model was then subjected to rounds of CHARMm minimization, initially with constraints that were applied to the proteinase; the co-factor was allowed to move freely. Later rounds were performed to convergence with no constraints. Dihedral constraints were applied to four residues in non-allowed conformations. A Ramachandran plot of the final model indicated that all residues were in allowed conformations.
We examined the positions of the mutations described in Fig. 1 with respect to the interactions seen in the crystal structure of the DEN-2 NS3 proteinase. In addition, the model between the NS3 proteinase and the NS2B co-factor was used to analyse three groups of mutations that mapped to the NS2B-binding cleft.
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Results |
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The first 181 amino acids of NS3 were scanned for clusters of five residues that contained at least three charged amino acids. Five such clusters outside proteinase motifs were chosen for mutagenesis and the charged residues were changed to alanine (Fig. 1) (Bass et al., 1991
). The changes made in DEN-2 NS3 were as follows: E17A, E19A and D20A; K63A, R64A and E66A; E91A, E93A and E94A; E169A, K170A and E173A; and lastly E179A, D180A and D181A. Alanine was chosen as the replacement amino acid since it removes the side chain beyond the
-carbon and also minimizes any steric effects within the polypeptide caused by the replacement (Cunningham & Wells, 1989
). In addition, two hydrophobic regions were chosen (Fig. 1
) on the basis of hydropathy plot data (Hahn et al., 1988
) and conservation of sequence across the flaviviruses (Westaway & Blok, 1997
; Chang, 1997
). They were G32A and Y33A, and V95A and Q96A. Thus a total of seven sites were mutated.
Transient expression and proteolysis of NS2B/3
The first experiments were designed to test for proteinase activity of mutant NS2B/3 proteins using transient expression of the pSV constructs in COS cells. We did not wish to investigate virus replication with mutations that abolished proteinase activity. Five constructs were tested. COS cells were transfected with the pSV plasmids listed in Fig. 1, radiolabelled and analysed by radioimmunoprecipitation and electrophoresis (Fig. 2
). As reported previously (Teo & Wright, 1997
), in cells transfected with the parental construct S2 and radiolabelled for 1 h at 37 °C, several polypeptides indicative of proteinase activity were detected using anti-NS3 antiserum (Fig. 2a
, lane 3). They were uncleaved NS2B/3 (83 kDa) and the cleavage products NS3 (69 kDa), NS2B/3' (64 kDa), NS3' (50 kDa) and NS3' (19 kDa). The bands corresponding to NS2B/3' and NS3' were faint and not well-resolved from host proteins; however, bands of NS2B/3, NS3 and NS3' were clear and sufficient for the assessment of proteolysis. NS2B (14 kDa) was detected by co-precipitation using anti-NS3 antiserum, as described for dengue virus and other flaviviruses (Arias et al., 1993
; Chambers et al., 1993
; Jan et al., 1995
; Teo & Wright, 1997
). The number and sizes of the observed proteins demonstrated that cleavage was occurring at the NS2B/NS3 and NS3'/NS3' sites. Proteinase activity was also detected in cells transfected by S1720, S3236, S6366 or S179181 and maintained at 37 °C (Fig. 2a
, lanes 46, 8). However, little or no cleavage occurred in cells transfected with the construct S9596; only NS2B/3 was readily detected (Fig. 2a
, lane 7).
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For all mutants shown in Fig. 2, with the exception of S9596, NS2B was co-precipitated with NS3 by anti-NS3 antiserum. The band corresponding to this protein was faint for S1720 (Fig. 2
, lanes 4), but was readily seen on longer exposure. Thus for these mutants, interaction between NS2B and NS3 was retained, consistent with the retention of proteinase activity.
Analysis of virus replication
Previous experiments showed that mutations that abolished or strongly reduced NS2B/3 proteinase activity usually prevented or greatly reduced virus replication (Nestorowicz et al., 1994 ; Chambers et al., 1993
; Amberg & Rice, 1999
), whereas mutations that retained activity generally allowed the recovery of infectious virus, albeit with reduced plaque titres and small plaque phenotypes (Nestorowicz et al., 1994
; Chambers et al., 1995
; Amberg & Rice, 1999
). Hence five mutations were chosen for incorporation into genomic-length cDNA and examination of their effects on virus replication. All three of the charged-to-alanine mutants tested (S1720, S6366 and S179181) did not show severe inhibition of proteinase activity in COS cells. We selected the mutations in two of these (S6366 and S179181) for incorporation into genomic-length cDNA and added a further two of the charged-to-alanine-type mutations (Fig. 1
) without prior testing in COS cells. For the mutations within hydrophobic regions (S3236 and S9596), only the changes of S3236 (cleavage of NS2B/3 detected) were incorporated into genomic-length cDNA.
Virus was produced from genomic-length cDNA by established procedures (Gualano et al., 1998 ). RNA was transcribed and electroporated into BHK-21 cells and the cells were incubated at 33 °C or 37 °C. BHK-21 cells were tested for immunofluorescence with anti-E antibodies. Medium from the transfected BHK-21 cells was passaged twice in C6/36 cells at 28 °C and virus titre was determined after the second passage by plaque assay in C6/36 cells. Viral RNA was then amplified by RTPCR and the complete NS2B and NS3 genes were sequenced to check that the mutation was retained during passaging and that no other base substitutions were introduced. Virus was derived at least twice for each construct and the results are summarized in Table 1
. Virus titres were determined for each experiment and the mean±SD corresponding to each construct is shown. Similar results were obtained consistently for a given construct.
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Growth of viruses V2, V6366, V169173 and V179181 in C6/36 and BHK-21 cells
C6/36 cells were infected with viruses V2, V6366, V169173 or V179181 at an m.o.i. of 1, maintained at 28 °C and the medium was sampled at 24 h intervals up to 96 h post-infection. Virus titres were determined by plaque assay in C6/36 cells. The resulting curves of released virus are shown in Fig. 4. The two viruses with the smallest plaque size (V6366 and V179181, Table 1
) initially lagged in virus release, although by 48 h after infection, their titres had reached from 1 to 8x104 p.f.u./ml, and by 72 h after infection, the yields of all four viruses were comparable (Fig. 4
). The observed delay in virus release for V6366 and V179181 was consistent with their very small plaque size. The presence of the respective mutations in the recovered viruses was confirmed by RTPCR and sequencing.
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Modelling the interaction of NS3 and NS2B
The 40 amino acid segment of NS2B required for the activity of the NS2B/3 proteinase is, overall, hydrophilic (L53E92) and shares no significant similarity with a protein of known structure. It contains a central hydrophobic region G69SSPILSITISE80 (Falgout et al., 1993 ; Brinkworth et al., 1999
), which was identified as the probable homologue to the HCV NS4A peptide in the HCV NS3/4A proteinase and was used to construct an homology model of the DEN-2 NS3/2B proteinase (Brinkworth et al., 1999
). The homology model was based on the structure of the HCV complex of NS3 (N-terminal 179 amino acids) and NS4A (peptide G21R34) (Yan et al., 1998
). However, an improved model for DEN-2 NS3/2B is now possible (Fig. 5
) using the coordinates for the crystal structure of the N-terminal 185 amino acids of DEN-2 NS3 (Murthy et al., 1999
). This model is useful for analysing the interactions between the NS2B peptide G69E80 and NS3, but further predictions on the effect of NS2B binding to the substrate-binding cleft or any direct interactions between NS2B and substrate would be overspeculative until the X-ray crystal structure of the NS2B/3 complex is determined.
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Discussion |
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Of the five charged sites mutated to alanine in DEN-2 NS3, three were examined by transient expression in COS cells, four were tested for their effects on virus replication and three were tested by both methods. For no mutant tested was proteinase activity or virus production abolished. Mutations related, but not identical, to those at E17LED20, K63RIE66, E91GEE94 and E169KSIE173 were tested for their effect on the activity of YFV NS2B/3 proteinase (Droll et al., 2000 ). No mutation reduced enzyme activity significantly except for those at E21D22 (YFV numbering). However, for the DEN-2 mutants, the yield of virus V9194, and to a lesser extent V6366 (Table 1
), were reduced compared with V169173 and V179181 and the parental virus V2. All mutant viruses showed reduced plaque size (Table 1
, Fig. 3
). The locations of the five charged sites were then mapped into a model of NS2B/3 (Fig. 5
). The model is based on the crystal structure of DEN-2 NS3 proteinase (Murthy et al., 1999
) and a fragment of NS2B corresponding to the fragment of NS4A seen in the structure of HCV proteinase (Brinkworth et al., 1999
).
The X-ray crystal structure of DEN-2 NS3 reveals that region E91GEE94, mutated in the low-yielding virus V9194, does not form part of the active-site cleft, nor does it interact with the fragment of NS2B in the model. E91 and E93 form salt bridges to R107. The loss of two salt bridges in the A91GAA94 mutation would be predicted to have a deleterious effect upon proteinase stability and possibly virus yield. However, the Q96 residue (see below) does form part of the predicted NS2B-binding cleft and we cannot exclude the possibility that E91GEE94 interacts with full-length NS2B. In HCV, the loops equivalent to residues 9094 and 140145 in DEN-2 NS3 are linked by interactions with a zinc ion. Interestingly, in the DEN-2 NS3 structure, the primary interaction between these loops is a hydrogen bond between the carbonyl oxygen of E94 and the side chain of K142. These data conflict with the prediction of Brinkworth et al. (1999) that E93 forms a salt bridge with K145. However, this prediction was based on an homology model developed from the HCV NS3 protein X-ray crystal structure before the availability of the DEN-2 NS3 proteinase structure.
The residues K63RIE66 (virus V6366) and E17LED20 (mutation not tested in virus) are both located at the N-terminal end of the NS2B-binding cleft. K63, R64 and E66 are solvent-exposed residues located at one end of the NS2B-binding cleft. The model of the NS3NS2B complex predicts that R64 makes a hydrogen bond to the carbonyl oxygen of the first residue in the NS2B peptide. We predict that more extensive interactions between R64 and E66 may be made with the full-length NS2B protein. The disruption of any one of these interactions, either alone or in combination, may explain the observed reduction in yield of virus V6366. The residues E17LED20 line the N-terminal end of the NS2B-binding cleft. We predict that E17 directly interacts with the carbonyl oxygen of G1 in the NS2B peptide.
The residues E169KSIE173 and E179DD181 lie at the C terminus of the proteinase at its junction with the helicase domain of NS3. Both sites are excluded from the minimal proteinase domain, defined as the N-terminal 167 amino acids of NS3 (Li et al., 1999 ) using in vitro transcription and translation. Residues E169KSIE173 form an
-helix (Fig. 5
) and the individual residues form hydrogen bonds with solvent molecules, apart from K170, which forms a hydrogen bond to the carbonyl oxygen of E167. These residues are located at the end of the substrate-binding cleft (on the P side; Schechter & Berger, 1967
) and thus may be important for determining substrate specificity. For E179DD181, the structure of NS3 reveals that D180 forms a hydrogen bond to the side chain of W69. The interaction with W69 is of particular interest, as this residue is located six residues N-terminal to the catalytic D75. Disruption of this hydrogen bond by the introduction of an alanine at position 180 may affect the conformation of the
-strand containing the catalytic aspartic acid and thus may impair proteinase activity.
In addition to the mutagenesis of the five charged sites, substitutions were made in two hydrophobic regions, G32YSQI36 and V95Q96. The residues G32 and V95Q96 lie outside the enzyme motifs but are highly conserved in members of the genus Flavivirus (Chang, 1997 ). The protein S3236 had autocatalytic activity (Fig. 2
, lanes 4), but the yield of V3236 was the lowest noted. G32 and Y33 line the N-terminal end of the NS2B-binding cleft. Interestingly, the crystal structure of NS3 reveals that Y33 bridges across the cleft, forming a hydrogen bond to the carbonyl oxygen of P10. We would expect a mutation at this position to affect NS2B binding. G32 forms part of a pocket that contains S3 from the NS2B peptide. We predict that mutation of this residue will affect the size of this pocket.
The mutations at V95Q96 are of particular interest as they are located at the C terminus of the NS2B-binding cleft. Substitution of these residues by alanine severely reduced self-cleavage of the S9596 protein (Fig. 2, lanes 7). Examination of the structure of NS3 reveals that V95 is buried in the hydrophobic core of the proteinase and that Q96 is solvent-exposed. In our model of NS3 complexed with the NS2B peptide, Q96 is directly beneath the C terminus of the NS2B peptide (Fig. 5
) and forms part of the binding cleft. The inability of the S9596 protein to self cleave suggests that mutation of these residues may affect the pre-cleavage interaction between NS3 and the NS2B co-factor and prevent proper processing at the NS2B/NS3 cleavage site.
Overall, the seven mutated sites were distributed evenly over the primary sequence of the NS3 proteinase and represented distinct regions in the model of NS3 complexed with the NS2B co-factor peptide. Of the mutations located, three (E17LED20, K63RIE66 and G32YSQI36) were at the N terminus of the NS2B-binding cleft, one (V95Q96) was at the C terminus of the cleft, two (E169KSIE173 and E179DD181) were at the C terminus of the proteinase domain and one (E91GEE94) was solvent-exposed. Thus, two of the charged regions (E17LED20 and K63RIE66) were adjacent to the NS2B-binding cleft. At present, it is unknown whether any of the other three charged regions interact with full-length NS2B. It is also possible that the basis for their effect on virus replication is unrelated to proteinase activity and may lie, for example, in the interaction of NS3 with other viral proteins such as NS5 (Kapoor et al., 1995 ; Chen et al., 1997
). Substitutions to alanine in conserved hydrophobic regions were more disruptive to self-cleavage (protein S9596) and virus production (virus V3236) than to changes in charged regions. A total of five viruses with reduced plaque size on C6/36 cells was obtained; two of these, V3236 and V9194, were possibly temperature-sensitive but did not grow sufficiently well for adequate testing (Table 1
). The remaining three viruses grew to reach good titres (Fig. 4
) and displayed small plaques but did not show the temperature-sensitive phenotype that has been observed for some viruses with charged-to-alanine mutations in non-structural genes (Diamond & Kirkegaard, 1994
; Parkin et al., 1996
; Muylaert et al., 1997
; Huang et al., 1998
; Gavin et al., 1999
). Virus V3236 replicated too poorly to be of further use and therefore the viruses of most interest with respect to growth restriction were V6366 and V9194. Both viruses contained mutations in charged amino acids, showed small plaque phenotypes and replicated less well than parental virus (V6366 only marginally less). The mutations contained in these viruses may be suitable for incorporation into growth-restricted vaccine strains. It may be possible to enhance the yield of V9194 by reducing the number of charged residues changed to alanine in the sequence E91GEE94 while retaining some growth restriction and a small plaque phenotype.
The results demonstrate that charged-to-alanine mutagenesis may be useful for obtaining growth-restricted viruses of other flavivirus species, either with mutations in the proteinase region or perhaps in other non-structural proteins. In our studies with DEN-2 NS3, we recovered infectious virus for all four of the charged-to-alanine mutants tested and the viruses displayed a useful range of growth restriction. Comparisons of the deduced amino acid sequences of flaviviruses show high conservation of hydrophilicity across the viral polyprotein, regardless of the considerable variation in primary sequence (Westaway & Blok, 1997 ) and thus it may be possible to extend these results to the other dengue virus serotypes and encephalitic flaviviruses. The mutations that were introduced here required multiple nucleotide and codon changes. In theory, multiple changes reduce the risk of reversion to parental phenotype when introduced into a potential vaccine strain. However, it would be necessary initially to assess each amino acid mutated in a cluster for its contribution to the mutant phenotype. The preferred situation is for each amino acid to make some contribution, rather than for one to be dominant.
The model of the NS3 complexed with an NS2B peptide co-factor enabled the definition of some individual residues important in the interaction between the two proteins. We predict that substitutions of these residues by amino acids other than alanine, both individually and in clusters, will confirm these interactions and expand our understanding of the flavivirus proteinase.
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Acknowledgments |
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References |
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Received 14 December 2000;
accepted .