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Short Communication |
Unité de Virologie Tropicale, Institut de Médecine Tropicale du Service de Santé des Armées (IMTSSA), BP 46, 13998 Marseille Armées, France
Correspondence
Boris A. M. Pastorino
publi.viro{at}laposte.net
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ABSTRACT |
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MAIN TEXT |
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The flavivirus genome consists of a positive- and single-stranded RNA of approximately 10 700 bases in length that contains only one functional open reading frame (ORF). This ORF is initially translated as a single polyprotein precursor, from which three structural proteins (capsid, membrane and envelope) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are produced by co- and post-translational cleavages. Cleavages at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 junctions depend on a virus-encoded protease (Falgout et al., 1991
). Being critical for virus replication, this protease is an interesting target for inhibitors with possible use as antiviral drugs.
Previous studies have located the flavivirus protease activity in the NS3 protein, whose 180 aa N-terminal domain, called NS3pro, exhibits the conserved catalytic triad of trypsin-like serine proteases (Bazan & Fletterick, 1989; Gorbalenya et al., 1989). Association of NS3pro with NS2B as a cofactor has been shown to be necessary for full activity of the protease against the different cleavage sites on the viral polyprotein. Several studies have demonstrated that a short (40 aa), hydrophilic, peptidic stretch of NS2B, called 
NS2B, is necessary and sufficient for this activity (Chambers et al., 1993; Falgout et al., 1991, 1993). Accordingly, two different strategies could be envisaged in order to inhibit the viral protease: one could target the catalytic domain and function, whilst another could consist of developing small compounds inhibiting interactions between NS2B and NS3. This kind of inhibitor could be highly specific, potentially presenting very few adverse side effects. One important question is about the possibility to conceive inhibitors with enough specificity to the targeted mechanism, but with broad-spectrum activity against the proteases of several flaviviruses. To answer this question, it is necessary to identify the residues or motifs responsible for the interaction, both on NS2B and NS3, and to determine whether they are conserved or homologous in different viruses or groups of viruses. Based on sequence analysis or mutagenesis experiments, several residues in the DENV-2, DENV-4 and YFV NS2B proteins have previously been shown to be critical for NS3 protease activation (Brinkworth et al., 1999; Butkiewicz et al., 2000; Chambers et al., 1993; Droll et al., 2000; Niyomrattanakit et al., 2004). However, DENV and YFV belong to the Aedes mosquito-borne cluster and can be considered related by several aspects. So, the conservation of the patterns that were proved to be critical in NS2B–NS3pro interaction needs confirmation for other flaviviruses.
In a previous study, we reported the enzymic characterization of ALKV, a virus belonging to the tick-borne flavivirus cluster (Charrel et al., 2001; Zaki, 1997). A catalytically active ALKV NS2B–NS3pro protease has been expressed as a hexahistidine recombinant protein, and an in vitro protease assay using a p-nitroanilide substrate (BAPNA) has shown that the association of NS3 with NS2B is necessary for protease activity (Bessaud et al., 2005).
In the present work, in an attempt to identify conserved residues and motifs essential for the NS2B–NS3 protease activity, we first compared the ALKV NS2B sequence with the corresponding sequences of other flaviviruses. Then, we tested the protease activity of ALKV NS3 when associated with NS2B from a different flavivirus or with ALKV NS2B with mutation or deletion on particular residues. Some conserved motifs previously identified as essential in NS2B of mosquito-borne flaviviruses were targeted.
Amino acid sequences of ALKV, Langat virus (LGTV), DENV-2, DENV-3, DENV-4, YFV and WNV were obtained from GenBank (accession nos AF331718
[GenBank]
, P29837
[GenBank]
, AF208496
[GenBank]
, AY099337
[GenBank]
, AF326573
[GenBank]
, U17067
[GenBank]
and NC_001563
[GenBank]
, respectively). They were aligned by using CLUSTAL_X software (Thompson et al., 1997). Hydrophobicity profiles of NS2B proteins were generated by using the Vector NTI suite v. 7 (InforMax Inc.).
The NS2B sequence alignment (Fig. 1) analysis revealed that only a few residues (L50, W60, G68, G81 and E89) are conserved in the seven studied flaviviruses. Sequence variation was distributed regularly along the NS2B region, affecting all of the motifs previously described for NS3 protease activation. The comparison of the primary structure of the studied ALKV NS2B domain indicated that NS2B amino acid differences observed between the two tick-borne flaviviruses ALKV and LGTV were about 25 %, with no change on the side-chain classes except for E57N and V65T. They reached about 75 % between the tick-borne and the mosquito-borne viruses (DENV, YFV and WNV), with numerous changes on the side-chain classes. Surprisingly, NS2B motifs previously identified as essential for NS3 activation in mosquito-borne virus sequences (52ELKK55, 67ISGS70, 75LSITI79 and 89EEEE92) (Butkiewicz et al., 2000; Droll et al., 2000; Wu et al., 2003) were poorly conserved in the two tick-borne flavivirus sequences studied. Furthermore, a hydrophobic stretch of the mosquito-borne viruses (70GSSPILSTISE81 for DENV-2) (Brinkworth et al., 1999), located within the hydrophilic cofactor domain, was not present in the ALKV or LGTV sequences (data not shown). These data were indicative of the poor conservation of the NS2B cofactor sequence within members of the genus Flavivirus.
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. PCR amplification was then realized by using Pfu Turbo DNA polymerase (Stratagene). After purification with spin columns (Qiagen), the PCR products were cloned into pET-DEST42 vector, according to the specification of Gateway Cloning Technology (Invitrogen). Constructs used for preliminary characterization of the ALKV NS2B–NS3 protease by Bessaud et al. (2005) were already available. They encoded, respectively, the NS3 protease domain (NS3 S1–E171), an active NS2B–NS3pro complex (NS3pro domain fused with NS2B M48–A94 and L126–R131 regions) and the NS2B–NS3pro HDA complex, where the S138A mutation in the triad makes the protease inactive. Alanine substitutions were introduced in the NS2B sequence at residues W60, G68, L73, Q77 and G81 by site-directed mutagenesis using the ‘megaprimer’ PCR method (Ke & Madison, 1997). In addition, substitution mutants V88D and V88K were generated, as well as a deletion mutant called V88. Two chimeric proteases were constructed, with the NS2B cofactor from LGTV and DENV-3 replacing the ALKV sequence. The two fused domains from LGTV and DENV-3 had previously been shown to activate NS3pro when used in homologous complexes (Bessaud et al., 2006). All 29 kDa mutated proteins were expressed, purified and analysed in a protease assay described previously (Bessaud et al., 2005). In previous experiments (Bessaud et al., 2006), we had observed precipitation of some flavivirus proteases in the presence of BAPNA. As a consequence, in all assays, we monitored in parallel A405 (for detection of BAPNA catalytic products) and A750 (indicative of turbidity variations). No substrate-dependent precipitation was detected for either ALKV mutants or chimeric proteins.
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NS2B–NS3pro (Table 2) showed that the LGT–ALK protease complex presented a low but detectable activity on BAPNA. Upon kinetic analysis, kcat and Km values were three- to fourfold less favourable than with the wild-type homologous protease. The LGTV NS2B cofactor seemed to affect both the turnover and the BAPNA affinity of the ALKV NS3 protease. This result could indicate a relative plasticity of the NS2B–NS3pro association, allowing partial activation of the protease by a heterologous cofactor. In contrast, the DEN3–ALK chimeric protease was totally inactive, suggesting that the few residues common to the ALKV and DENV-3 NS2B domains were not sufficient to promote a correct folding of NS3pro.
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NS2B sequence produced proteins retaining 18–77 % activity compared with that of the wild-type enzyme. In previous study, a different result had been reported for DENV-2, where alanine substitution at the conserved residue W62 led to negligible protease activity on fluorogenic peptide substrate (Niyomrattanakit et al., 2004).
In DENV, the motif called 
x3 (75LSITI79 for DENV-2) has been proposed to play a functional role in the association of the flavivirus proteases with their corresponding cofactors (Butkiewicz et al., 2000). It has also been shown that specific residues located within the structural x3 motif were important for activation of the protease activity (Niyomrattanakit et al., 2004). However our sequence comparison showed that for different viruses, including ALKV, LGTV and also DENV-4 and YFV, this x3 motif appeared poorly conserved with, in particular, the absence of one critical bulky, hydrophobic amino acid. Moreover, for ALKV, the x3 motif was represented by the sequence 73LKVRQ77 and alanine substitution at the position Q77 had a stronger effect on the activity of the NS3 protease than the same substitution at the position L73. Kinetic analysis showed that the mutation Q77A had greater effects on substrate binding than on the reaction rate; this result is not in agreement with a previously proposed model for NS2B-dependent activation of the DENV NS3 protease, where the cofactor contributes mainly to the arrangement of the residues in the catalytic pocket (Niyomrattanakit et al., 2004).
DENV-2 NS2B motif 89EEEE92 (Wu et al., 2003) or 89VEET92 motif for DENV-4 (Falgout et al., 1993) has been shown to be essential for the activity of the protease complex. For ALKV, this motif was represented by the sequence 88VEKE91 and, by analogy, the NS2B residue V88 was identified as crucial for the ALKV complex activity in our in vitro assay. Furthermore, charged amino acid substitutions at the same position (mutants V88D and V88K) resulted in an inactivation of the ALKV protease, with a 25- to 80-fold-reduced activity compared with that of the wild-type NS2B–NS3pro protease. These two replacements affected the kcat values of the recombinant proteases, with about a 30-fold reduction compared with the wild-type protease, indicating a rearrangement of the residues of the catalytic triad.
It was proposed that NS2B functioned as a molecular chaperone in assisting the folding of NS3pro to an active conformation (Leung et al., 2001), but the precise mechanism of cofactor-dependent activation was not elucidated. Recently, NS2B–NS3pro complex crystal structures of DENV-2 and WNV have been reported (Erbel et al., 2006). These structures indicated that WNV NS2B residues D82–F85 were involved in substrate recognition, whilst residues R78–L87 were involved in linking NS2B to NS3pro. However, for DENV, the crystal structures could not explain the absolute requirement of NS2B for NS3pro activity. Finally, the results obtained by Erbel et al. (2006) revealed some residues of NS2B important for the stabilization of the NS3pro active structure, but these residues were in part different from those that were identified previously. The significant differences between ALKV and WNV/DENV shown in this article would complicate modelling of the NS2B–NS3 complex based on sequence comparisons and the development of a single model applicable to both mosquito-borne and tick-borne flaviviruses. The residues of NS2B critical and strictly necessary for NS3 protease activation were not, until now, clearly identified. By analogy, our results seemed to show that the flavivirus protease-activation mechanism is complex and poorly conserved.
In conclusion, for the first time, the enzyme–cofactor interactions of a tick-borne flavivirus were analysed and two NS2B residues critical for ALKV NS3 activation, V88 and Q77, were identified. These results were compared with all others, and in vitro assays, realized by using recombinant proteases belonging to the mosquito-borne cluster and modifications in important NS2B motifs, showed some interesting differences. This study highlighted the need to extend the experimental identification of important NS2B residues with protease complexes belonging to different flavivirus clusters. In this way, future studies will concern the enzymic characterization of selective chimeric proteinases fusing NS2B and NS3pro domains from different flaviviruses. The construction and the analyses of some corresponding mutants will also be realized. Moreover, trans-activation assays combining NS3pro and NS2B expressed separately are in progress, as well as interaction tests with NS2B-specific synthetic peptides to clarify the understanding of the molecular NS2B–NS3 complex formation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTMAIN TEXTREFERENCES |
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Bessaud, M., Grard, G., Peyrefitte, C. N., Pastorino, B., Rolland, D., Charrel, R. N., de Lamballerie, X. & Tolou, H. J. (2005). Identification and enzymatic characterization of NS2B-NS3 protease of Alkhurma virus, a class-4 flavivirus. Virus Res 107, 57–62.[CrossRef][Medline]
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Butkiewicz, N., Yao, N., Zhong, W. & 10 other authors (2000). Virus-specific cofactor requirement and chimeric hepatitis C virus/GB virus B nonstructural protein 3. J Virol 74, 4291–4301.
Chambers, T. J., Nestorowicz, A., Amberg, S. M. & Rice, C. M. (1993). Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing NS2B-NS3 complex formation, and viral replication. J Virol 67, 6797–6807.
Charrel, R. N., Zaki, A. M., Attoui, H. & 7 other authors (2001). Complete coding sequence of the Alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in Saudi Arabia. Biochem Biophys Res Commun 287, 455–461.[CrossRef][Medline]
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Erbel, P., Schiering, N., D'Arcy, A. & 7 other authors (2006). Structural basis for the activation of flaviviral NS3 proteases from dengue and West Nile virus. Nat Struct Mol Biol 13, 372–373.[CrossRef][Medline]
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Falgout, B., Miller, R. H. & Lai, C.-J. (1993). Deletion analysis of dengue virus type 4 nonstructural protein NS2B: identification of a domain required for NS2B-NS3 protease activity. J Virol 67, 2034–2042.
Gorbalenya, A. E., Donchenko, A. P., Koonin, E. V. & Blinov, V. M. (1989). N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res 17, 3889–3897.
Ke, S.-H. & Madison, E. L. (1997). Rapid and efficient site-directed mutagenesis by single-tube ‘megaprimer’ PCR method. Nucleic Acids Res 25, 3371–3372.
Leung, D., Schroder, K., White, H., Fang, N.-X., Stoermer, M. J., Abbenante, G., Martin, J. L., Young, P. R. & Fairlie, D. P. (2001). Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors. J Biol Chem 276, 45762–45771.
Niyomrattanakit, P., Winoyanuwattikun, P., Chanprapaph, S., Angsuthanasombat, C., Panyim, S. & Katzenmeier, G. (2004). Identification of residues in the dengue virus type 2 cofactor that are critical for NS3 protease activation. J Virol 78, 13708–13716.
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Received 31 March 2006;
accepted 23 June 2006.
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